Co-reporter:Junfeng Huang;Hongqiang Qin;Jing Dong;Chunxia Song;Yangyang Bian;Mingming Dong;Kai Cheng;Fangjun Wang;Deguang Sun;Liming Wang;Mingliang Ye
Journal of Proteome Research September 5, 2014 Volume 13(Issue 9) pp:3896-3904
Publication Date(Web):2017-2-22
DOI:10.1021/pr500454g
Current sample preparation protocols for quantitative phosphoproteome analysis are tedious and time-consuming. Here, a facile in situ sample processing approach (iSPA) is developed by using macroporous Ti(IV)-IMAC microspheres as the preparation “beds”, where all sample preparation procedures including the enrichment of phosphoproteins, tryptic digestion of proteins, enrichment, and isotope labeling of phosphopeptides are performed in situ sequentially. As a result of the in situ processing design and the seamless procedures, extra steps for desalting and buffer exchanging, which are always required in conventional approaches, are avoided, and the sample loss and contamination could be greatly reduced. Thus, better sensitivity and accuracy for the quantitative phosphoproteome analysis were obtained. This strategy was further applied to differential phosphoproteome analysis of human liver tissues with or without hepatocellular carcinoma (HCC). In total, 8548 phosphorylation sites were confidently quantified from three replicate analyses of 0.5 mg of human liver protein extracts.Keywords: dimethyl labeling; hepatocellular carcinoma; human liver; in situ sample processing approach; on-beads digestion; phosphoprotein enrichment; phosphoproteome quantification; solid phase labeling;
Co-reporter:Houjiang Zhou, Mingliang Ye, Jing Dong, Guanghui Han, Xinning Jiang, Renan Wu and Hanfa Zou
Journal of Proteome Research September 5, 2008 Volume 7(Issue 9) pp:3957-3967
Publication Date(Web):July 17, 2008
DOI:10.1021/pr800223m
The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti4+-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti4+ through the chelating interaction between phosphate groups on the polymer and Ti4+. The resulting Ti4+-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti4+-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti4+-IMAC to other enrichment methods including Fe3+-IMAC, Zr4+-IMAC, TiO2 and ZrO2, and demonstrate superior selectivity and efficiency of Ti4+-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti4+-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.Keywords: Immobilized metal ion affinity chromatography; phosphate polymer; phosphopeptide enrichment; phosphoproteome analysis; titanium ion;
Co-reporter:Xinning Jiang;Mingliang Ye;Kai Cheng
Journal of Proteome Research May 7, 2010 Volume 9(Issue 5) pp:2743-2751
Publication Date(Web):Publication Date (Web): March 24, 2010
DOI:10.1021/pr9009904
The development of new phosphoproteomic technologies has led to a rapid increase in the number of phosphoprotein identifications. Managing and extracting valuable information from the phosphoproteome data sets and generating output information in user-friendly formats require special data management and process platform. Even though a few proteome pipelines have been developed, they are mainly designed for processing data set of unmodified peptide/protein identifications. Because of the different characteristics of phosphorylated peptides/proteins, these pipelines are inconvenient, sometimes inappropriate, to process the phosphoproteome data sets. In this study, a software suite named ArMone was specially designed for the management and analysis of phosphoproteome data. It can readily identify phosphopeptides with high reliability and high sensitivity, and can effectively pinpoint the most probable phosphorylation site. A few well-designed postvalidation process tools are also available to extract and export valuable information. ArMone is a stand-alone application with friendly graphic user interface. It can run on different operating systems and can process data sets obtained by most of the commonly used database search engines.Keywords: automatic validation; bioinformatics; phosphoproteome analysis; software;
Co-reporter:Deguang Sun;Fangjun Wang;Rui Chen;Xinning Jiang;Guanghui Han;Liming Wang;Mingliang Ye
Journal of Proteome Research February 6, 2009 Volume 8(Issue 2) pp:651-661
Publication Date(Web):Publication Date (Web): January 21, 2009
DOI:10.1021/pr8008012
The study of protein glycosylation has lagged far behind the progress of current proteomics because of the enormous complexity, wide dynamic range distribution and low stoichiometric modification of glycoprotein. Solid phase extraction of tryptic N-glycopeptides by hydrazide chemistry is becoming a popular protocol for the analysis of N-glycoproteome. However, in silico digestion of proteins in human proteome database by trypsin indicates that a significant percentage of tryptic N-glycopeptides is not in the preferred detection mass range of shotgun proteomics approach, that is, from 800 to 3500 Da. And the quite big size of glycan groups may block trypsin to access the K, R residues near N-glycosites for digestion, which will result in generation of big glycopeptides. Thus many N-glycosites could not be localized if only trypsin was used to digest proteins. Herein, we describe a comprehensive way to analyze the N-glycoproteome of human liver tissue by combination of hydrazide chemistry method and multiple enzyme digestion. The lysate of human liver tissue was digested with three proteases, that is, trypsin, pepsin and thermolysin, with different specificities, separately. Use of trypsin alone resulted in identification of 622 N-glycosites, while using pepsin and thermolysin resulted in identification of 317 additional N-glycosites. Among the 317 additional N-glycosites, 98 (30.9%) could not be identified by trypsin in theory because the corresponding in silico tryptic peptides are either too small or too big to detect in mass spectrometer. This study clearly demonstrated that the coverage of N-glycosites could be significantly increased due to the adoption of multiple enzyme digestion. A total number of 939 N-glycosites were identified confidently, covering 523 noredundant glycoproteins from human liver tissue, which leads to the establishment of the largest data set of glycoproteome from human liver up to now.Keywords: Glycoproteomics; Human liver proteome project; Hydrazide chemistry; Multiple enzyme digestion;
Co-reporter:Yixin Zhang, Yanbo Pan, Wujun Liu, Yongjin J. Zhou, Keyun Wang, Lei Wang, Muhammad Sohail, Mingliang Ye, Hanfa Zou and Zongbao K. Zhao
Chemical Communications 2016 vol. 52(Issue 40) pp:6689-6692
Publication Date(Web):14 Apr 2016
DOI:10.1039/C6CC02386J
An approach combining in vivo protein allylation, chemical tagging and affinity enrichment was devised to capture protein methylation candidates in yeast S. cerevisiae. The study identified 167 hits, covering many proteins with known methylation events on different types of amino acid residues.
Co-reporter:Fangjie Liu, Hao Wan, Zhongshan Liu, Hongwei Wang, Jiawei Mao, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2016 Volume 88(Issue 10) pp:5058
Publication Date(Web):April 21, 2016
DOI:10.1021/acs.analchem.6b00701
In this study, we developed a Ti(IV) monolithic spin tip for phosphoproteome analysis of a minute amount of biological sample for the first time. The surface of polypropylene pipet tip was activated by the photoinitiator benzophenone under UV light radiation followed by polymerization of ethylene glycol methacrylate phosphate and bis-acrylamide in the tip to form a porous monolith with reactive phosphate groups. The as-prepared tips grafted with monolithic adsorbent were then chelated with titanium(IV) ion for phosphopeptide enrichment. It was found that the tips enabled fast and efficient capture of phosphopeptides from microscale complex samples. The monolithic tip was demonstrated to have a detection limit as low as 5 fmol β-casein tryptic digest, along with an exceptionally high specificity to capture phosphopeptides from complex tryptic digest mixed with an unphosphorylated protein and a phosphorylated protein at a molar ratio up to 1000:1. When the tip was applied to enrich phosphopeptides from 5 μg of tryptic digest of complex HeLa cell proteins, 1185 high confidence of phosphorylated sites were successfully identified with the specificity as high as 92.5%. So far, this is the most sensitive phosphoproteomics analysis using a standard liquid chromatography–tandem mass spectrometry (LC–MS/MS) system for proteome-wide phosphorylation analysis in mammalian cells.
Co-reporter:Jingyao Bai, Hongwei Wang, Junjie Ou, Zhongshan Liu, Yehua Shen, Hanfa Zou
Analytica Chimica Acta 2016 Volume 925() pp:88-96
Publication Date(Web):21 June 2016
DOI:10.1016/j.aca.2016.04.012
•A highly crosslinked polymeric monolith was fast prepared with a multi-acrylate monomer as crosslinker within 5 min.•The addition of thiol eliminated the major obstacle of oxygen inhibition in the thiol-ene (acrylate/methacrylate) polymerization.•Poly(ODT-co-DPEPA) monolith exhibited efficient chromatographic performance for separation of alkylbenzenes and tryptic digest of proteins.A facile approach was exploited for fast preparation of polymer-based monoliths in UV-transparent fused-silica capillaries via “one-pot” photo-initiated thiol-acrylate polymerization reaction of dipentaerythritolpenta-/hexaacrylate (DPEPA) and 1-octadecanethiol (ODT) in the presence of porogenic solvents (1-butanol and ethylene glycol). Due to relative insensitivity of oxygen inhibition in thiol-ene free-radical polymerization, the polymerization could be performed within 5 min. The effects of composition of prepolymerization solution on the morphology and permeability of poly(ODT-co-DPEPA) monoliths were investigated in detail by adjusting the content of monomer and binary porogen ratio. The physical properties of poly(ODT-co-DPEPA) monoliths were characterized by Fourier transform infrared spectroscopy (FT-IR), mercury intrusion porosimetry (MIP) and nitrogen adsorption/desorption measurement. The evaluation of chromatographic performance was carried out by capillary liquid chromatography (cLC). The results indicated that the poly(ODT-co-DPEPA) monolith was homogeneous and permeable, and also possessed a typical reversed-phase retention mechanism in cLC with high efficiency (∼75,000 N m−1) for separation of alkylbenzenes. Eventually, the further separation of tryptic digest of proteins by cLC tandem mass spectrometry (cLC-MS/MS) demonstrated its potential in the analysis of biological samples.
Co-reporter:Hongwei Wang, Junjie Ou, Jingyao Bai, Zhongshan Liu, Yating Yao, Lianfang Chen, Xiaojun Peng, Hanfa Zou
Journal of Chromatography A 2016 Volume 1436() pp:100-108
Publication Date(Web):4 March 2016
DOI:10.1016/j.chroma.2016.01.063
•Thiol-acrylate photopolymerization was adopted to prepare poly(PEDAS-co-TPTM) column.•The column possessed bicontinuous structure.•The column exhibited better permeability (1.68 × 10−14 m2) than poly(PEDAS) column (9.2 × 10−15 m2).•The column exhibited lower plate heights (15.7–17.7 μm) than poly(PEDAS) column (19.1–37.9 μm).A simple approach was developed for rapid preparation of polymeric monolithic columns in UV-transparent fused-silica capillaries via photoinitiated thiol-acrylate polymerization of pentaerythritol diacrylate monostearate (PEDAS) and trimethylolpropane tris(3-mercaptopropionate) (TPTM) within 10 min, in which the acrylate homopolymerized and copolymerized with the thiol simultaneously. The morphology, permeability and chromatographic performance of the resulting poly(PEDAS-co-TPTM) monoliths were studied. It could be observed from SEM that the morphology of poly(PEDAS-co-TPTM) monolith was rather different from that of poly(PEDAS) monolith, which was fabricated via photo-induced free radical polymerization using PEDAS as the sole monomer. Compared with poly(PEDAS) monolith, poly(PEDAS-co-TPTM) monolith possessed better permeability when they were fabricated under the same preparation conditions. By adjusting the composition of porogenic solvents, poly(PEDAS-co-TPTM) monolith exhibited lower plate heights (15.7–17.7 μm) than poly(PEDAS) monolith (19.1–37.9 μm) in μLC. In addition, 66 unique peptides were positively identified on poly(PEDAS-co-TPTM) monolith when tryptic digest of four proteins was separated by μLC-MS/MS, demonstrating its potential in proteome analysis.
Co-reporter:Yan Jin, Yang Yu, Yanxia Qi, Fangjun Wang, Jiaze Yan, Hanfa Zou
Journal of Proteomics 2016 Volume 141() pp:24-46
Publication Date(Web):1 June 2016
DOI:10.1016/j.jprot.2016.04.010
•The numbers of identified peptides were 250 in yogurt, 434 in gastric digest and 466 in pancreatic digest.•Gastric and pancreatic digests presented the better ACE and DPP-IV inhibition activity than yogurt.•κ-CN contributed the diversity of peptides in fermentation processing and β-CN did it in gastrointestinal digestion.This study investigated the relationship between peptide profiles and the bioactivity character of yogurt in simulated gastrointestinal trials. A total of 250, 434 and 466 peptides were identified by LC-MS/MS analyses of yogurt, gastric digest and pancreatic digest. Forty peptides of yogurt survived in gastrointestinal digestion. κ-CN and β-CN contributed the diversity of peptides during the fermentation process and gastrointestinal digestion, respectively. The favorite of κ-CN by lactic acid bacteria complemented gut digestion by hydrolyzing κ-CN, the low abundance milk proteins. The potential bioactivities were evaluated by in vitro ACE and DPP-IV inhibition assays. The ACE inhibition rate of the pancreatic digests was ~ 4 - and ~ 2 - fold greater than that of yogurt and the gastric digests. The ACE inhibitory peptides generated during gastrointestinal digestion improved the ACE inhibitory activity of the gastric and pancreatic digests. The DPP-IV inhibition rate of the pancreatic digest was ~ 6 - and ~ 3 - fold greater than that of yogurt and the gastric digest. The numbers of potential DPP-IV inhibitory peptides were positively correlated to the DPP-IV inhibitory activity of the gastric and pancreatic digests.Biological significanceThe present study describes the characters and bioactivities of peptides from yogurt in a simulated gastrointestinal digestion. The number of peptides identified from yogurt and gastrointestinal digests by LC-MS/MS increased in the simulated gastrointestinal trials. The in vitro ACE and DPP-IV inhibition bioactivities revealed that the bioactivity of yogurt was enhanced during gastrointestinal digestion. The correlation between peptides and bioactivity in vitro indicated that not only the peptides amount but also the proportion of peptides with high bioactivities contributed to increased bioactivity during gastrointestinal digestion. The study of peptides identified from yogurt and digests revealed that the number of released peptides was not determined by the abundance of the parent proteins but by whether the enzymes favored the protein.In summary, peptide profiling and bioactivities of yogurt in simulated gastrointestinal digestion helped to elucidate the health benefits of yogurt peptides. The results further revealed that pre-digestion of milk by lactic acid bacteria are complementary to generate bioactive peptides and to provide particular yogurt functions.
Co-reporter:Quanqing Zhang, Qinghe Zhang, Zhichao Xiong, Hao Wan, Xiaoting Chen, Hongmei Li, Hanfa Zou
Talanta 2016 Volume 146() pp:272-278
Publication Date(Web):1 January 2016
DOI:10.1016/j.talanta.2015.08.068
•A sandwich-like composite composed of ordered mesoporous carbon–silica shell-coated graphene was prepared.•The carbon–silica shell was synthesized by in-situ carbonization.•A great performance has been shown that the material could capture lots of peptides from bovine serum albumin.•The material could efficiently size-exclude proteins and enriches the low-abundant peptides in human serum.A sandwich-like composite composed of ordered mesoporous carbon–silica shell-coated graphene (denoted as graphene@mSiO2-C) was prepared by an in-situ carbonation strategy. A mesoporous silica shell was synthesized by a sol–gel method, and cetyltrimethyl ammonium bromide inside the mesopores were in-situ carbonized as a carbon source to obtain a carbon–silica shell. The resulting mesoporous carbon–silica material with a sandwich structure possesses a high surface area (600 m2 g−1), large pore volume (0.587 cm3 g−1), highly ordered mesoporous pore (3 nm), and high carbon content (30%). This material shows not only high hydrophobicity of graphene and mesoporous carbon but also a hydrophilic silica framework that ensures excellent dispersibility in aqueous solution. The material can capture many more peptides from bovine serum albumin tryptic digests than mesoporous silica shell-coated graphene, demonstrating great enrichment efficiency for peptides. Furthermore, the prepared composite was applied to the enrichment of low-abundance endogenous peptides in human serum. Based on Matrix-Assisted Laser Desorption/ Ionization Time of Flight Mass Spectrometry identification, the graphene@mSiO2-C could efficiently size-exclude proteins and enriches the low-abundant peptides on the graphene and mesoporous carbon. And based on the LC-MS/MS results, 892 endogenous peptides were obtained by graphene@mSiO2-C, hinting at its great potential in peptides analysis.The synthesis procedure of graphene@mSiO2-C used for the selective enrichment of endogenous peptides.
Co-reporter:Zhongshan Liu, Junjie Ou, Hanfa Zou
TrAC Trends in Analytical Chemistry 2016 Volume 82() pp:89-99
Publication Date(Web):September 2016
DOI:10.1016/j.trac.2016.05.016
•Click polymerization was used for direct construction of monolithic columns.•Monolithic columns can generate plate height with smaller than 10 µm in LC.•The phase separation process of click polymerization was discussed.In recent years, polymerizations based on click reactions (thiol-ene, thiol-yne, thiol-Michael, thiol-epoxy and amine-epoxy) have been utilized to prepare monolithic columns. These polymerization systems are easily carried out under mild conditions. Either hybrid or organic monolithic columns fabricated by click polymerization demonstrated homogeneous network structures. For separation of small molecules, the column efficiencies, such as plate height with less than 10 µm, have been greatly improved comparing with organic monolithic columns prepared with free radical polymerization. In this review, we will summarize recent progress on the preparation of monolithic columns and their chromatographic performances.
Co-reporter:Yajing Chen, Zhichao Xiong, Lingyi Zhang, Jiaying Zhao, Quanqing Zhang, Li Peng, Weibing Zhang, Mingliang Ye and Hanfa Zou
Nanoscale 2015 vol. 7(Issue 7) pp:3100-3108
Publication Date(Web):30 Dec 2014
DOI:10.1039/C4NR05955G
Highly selective and efficient capture of glycosylated proteins and peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. Recently, hydrophilic interaction liquid chromatography (HILIC)-based functional materials have been extensively utilized for glycopeptide enrichment. However, the low amount of immobilized hydrophilic groups on the affinity material has limited its specificity, detection sensitivity and binding capacity in the capture of glycopeptides. Herein, a novel affinity material was synthesized to improve the binding capacity and detection sensitivity for glycopeptides by coating a poly(2-(methacryloyloxy)ethyl)-dimethyl-(3-sulfopropyl) ammonium hydroxide (PMSA) shell onto Fe3O4@SiO2 nanoparticles, taking advantage of reflux-precipitation polymerization for the first time (denoted as Fe3O4@SiO2@PMSA). The thick polymer shell endows the nanoparticles with excellent hydrophilic property and several functional groups on the polymer chains. The resulting Fe3O4@SiO2@PMSA demonstrated an outstanding ability for glycopeptide enrichment with high selectivity, extremely high detection sensitivity (0.1 fmol), large binding capacity (100 mg g−1), high enrichment recovery (above 73.6%) and rapid magnetic separation. Furthermore, in the analysis of real complicated biological samples, 905 unique N-glycosylation sites from 458 N-glycosylated proteins were reliably identified in three replicate analyses of a 65 μg protein sample extracted from mouse liver, showing the great potential of Fe3O4@SiO2@PMSA in the detection and identification of low-abundance N-linked glycopeptides in biological samples.
Co-reporter:Hao Wan, Yi Zhang, Weibing Zhang, and Hanfa Zou
ACS Applied Materials & Interfaces 2015 Volume 7(Issue 18) pp:9608
Publication Date(Web):April 20, 2015
DOI:10.1021/acsami.5b01165
In consideration of the intrinsic complexity of cancer, just being a delivery nanovehicle for the nanocarrier is no longer enough to fulfill requirements of dealing with cancer. In this regard, the multifunctional nanocarrier appears to be an appealing choice in cancer treatment. Herein, the novel multifunctional nanocarrier (Fe3O4-NS-C3N4@mSiO2-PEG-RGD) possessing properties of dual targeting (the peptide- and magnetism-mediated targeting), imaging (one- and two-photon modes), pH-triggered release of loaded anticancer drug, and synergistic treatment (photodynamic therapy (PDT) combined with chemotherapy) are successfully developed. The nanocarrier specifically centralizes within cancer cells with the enhanced amount through the dual targeting ability and is facilely tracked under one- and two-photon imaging modes attributed to the autofluorescence. Then, visible light irradiation-induced PDT combined with low pH-triggered chemotherapy synergistically cooperate to efficiently kill cancer cells. Following the above process, the multifunctional nanocarrier demonstrates effective inhibition of the growth of A549 and HeLa cancer cells. The efficient manipulation of Fe3O4-NS-C3N4@mSiO2-PEG-RGD also implies potential applications of the multifunctional nanocarrier in delivery of different agents. Furthermore, it might also broaden the scope of fabrication of the multifunctional nanocarrier for inhibiting the growth of cancer cells.Keywords: chemo-photodynamic synergistic treatment; dual targeting; multifunctional nanocarrier; pH-triggered release; two-photon imaging;
Co-reporter:Jin Chen, Zheyi Liu, Fangjun Wang, Jiawei Mao, Ye Zhou, Jing Liu, Hanfa Zou and Yukui Zhang
Chemical Communications 2015 vol. 51(Issue 79) pp:14758-14760
Publication Date(Web):13 Aug 2015
DOI:10.1039/C5CC06072A
We develop an acidic vapor assisted electrospray ionization strategy within an enclosed electrospray ionization source to counteract the ion suppression effects caused by trifluoroacetic acid. The mass spectrometry signal intensity of intact proteins was improved 10 times and the number of valid signals for E. coli intact protein samples was improved 96% by using this strategy.
Co-reporter:Hao Wan, Junfeng Huang, Zhongshan Liu, Jinan Li, Weibing Zhang and Hanfa Zou
Chemical Communications 2015 vol. 51(Issue 45) pp:9391-9394
Publication Date(Web):30 Apr 2015
DOI:10.1039/C5CC01980J
Combining high surface area, numerous active sites, strong magnetic response, multivalent synergistic binding effects and outstanding hydrophilicity, a novel hydrophilic composite demonstrated efficient and selective enrichment of glycopeptides from the complex sample.
Co-reporter:Jinan Li, Fangjun Wang, Jing Liu, Zhichao Xiong, Guang Huang, Hao Wan, Zheyi Liu, Kai Cheng and Hanfa Zou
Chemical Communications 2015 vol. 51(Issue 19) pp:4093-4096
Publication Date(Web):03 Feb 2015
DOI:10.1039/C5CC00187K
Magnetic nanoparticles functionalized with maltosylated glycopeptide dendrimers were prepared via azide-alkynyl click reaction. The functionalized magnetic nanoparticles exhibited high hydrophilicity and good efficiency in glycopeptide enrichment by HILIC.
Co-reporter:Tian Jin, Zhichao Xiong, Xiang Zhu, Nada Mehio, Yajing Chen, Jun Hu, Weibing Zhang, Hanfa Zou, Honglai Liu, and Sheng Dai
ACS Macro Letters 2015 Volume 4(Issue 5) pp:570
Publication Date(Web):May 1, 2015
DOI:10.1021/acsmacrolett.5b00235
A facile template-free strategy for the synthesis of mesoporous phenolic polymers with attractive porosities, nitrogen-containing functionalities, and intrinsic hydrophilic skeletons is presented. The resultant polymer has a high BET surface area (548 m2 g–1) and mesopore size (13 nm) and exhibits superior glycopeptide-capturing performance, thus, revealing the potential application of mesoporous polymers in highly selective glycopeptide enrichment. This general capture protocol may open up new opportunities for the development of glycoproteomes.
Co-reporter:Jing Liu, Fangjun Wang, Jiawei Mao, Zhang Zhang, Zheyi Liu, Guang Huang, Kai Cheng, and Hanfa Zou
Analytical Chemistry 2015 Volume 87(Issue 4) pp:2054
Publication Date(Web):February 3, 2015
DOI:10.1021/ac504700t
N-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain the enzyme activity and solubilize hydrophobic proteins without changing their structures, was applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least than those obtained by conventional strategies.
Co-reporter:Junfeng Huang, Hao Wan, Yating Yao, Jinan Li, Kai Cheng, Jiawei Mao, Jin Chen, Yan Wang, Hongqiang Qin, Weibing Zhang, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2015 Volume 87(Issue 20) pp:10199
Publication Date(Web):September 23, 2015
DOI:10.1021/acs.analchem.5b02669
Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.
Co-reporter:Yating Yao, Junfeng Huang, Kai Cheng, Yanbo Pan, Hongqiang Qin, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2015 Volume 87(Issue 22) pp:11353
Publication Date(Web):October 15, 2015
DOI:10.1021/acs.analchem.5b02711
A problem for “shot-gun” proteomics is that the peptides generated in the proteolysis step overwhelm the analytical capacity of current LC–MS/MS systems. A straightforward approach to overcome this problem is to reduce the sample complexity by isolating the representative peptides of each protein. In this study, we presented a facile solid-phase capture-release approach to selectively enrich the peptides with N-terminal serine/threonine from protein digests. This method exploited the highly efficient reaction between an aldehyde group and a hydrazine group. The excellent performance of this approach was validated using synthetic peptides as well as complex protein digests. It was found that high enrichment specificity could be obtained and the identifications for complex samples with and without enrichment were highly complementary. Besides, the enrichment of peptides with serine/threonine adjacent to different protease cleavage sites demonstrated that our method was able to enrich peptides from protein digests in a sequence specific way. As a result, this new approach provides a simple way to reduce sample complexity and facilitates the identification of low-abundance proteins.
Co-reporter:Haiyang Zhang, Junjie Ou, Zhongshan Liu, Hongwei Wang, Yinmao Wei, and Hanfa Zou
Analytical Chemistry 2015 Volume 87(Issue 17) pp:8789
Publication Date(Web):July 30, 2015
DOI:10.1021/acs.analchem.5b01707
A novel “one-pot” approach was developed for ultrarapid preparation of various hybrid monolithic columns in UV-transparent fused-silica capillaries via photoinitiated thiol–acrylate polymerization of an acrylopropyl polyhedral oligomertic silsesquioxane (acryl-POSS) and a monothiol monomer (1-octadecanethiol or sodium 3-mercapto-1-propanesulfonate) within 5 min, in which the acrylate not only homopolymerizes, but also couples with the thiol. This unique combination of two types of free-radical reaction mechanisms offers a simple way to fabricate various acrylate-based hybrid monoliths. The physical characterization, including scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and thermal gravimetric analysis was performed. The results indicated that the monothiol monomers were successfully incorporated into acryl-POSS-based hybrid monoliths. The column efficiencies for alkylbenzenes on the C18-functionalized hybrid monolithic column reached to 60 000–73 500 plates/m at the velocity of 0.33 mm/s in capillary liquid chromatography, which was far higher than that of previously reported POSS-based columns prepared via thermal-initiated free-radical polymerization without adding any thiol monomers. By plotting the plate height (H) of the alkylbenzenes versus the linear velocity (u) of the mobile phase, the results revealed a retention-independent efficient performance of small molecules in the isocratic elution. These results indicated that more homogeneous hybrid monoliths formed via photoinitiated thiol–acrylate polymerization; particularly, the use of the multifunctional cross-linker possibly prevented the generation of gel-like micropores, reducing mass transfer resistance (C-term). Another sulfonate-containing hybrid monolithic column also exhibited hydrophobicity and ion-exchange mechanism, and the dynamic binding capacity was calculated as 71.1 ng/cm (75 μm i.d.).
Co-reporter:Hui Lin, Junjie Ou, Zhongshan Liu, Hongwei Wang, Jing Dong, and Hanfa Zou
Analytical Chemistry 2015 Volume 87(Issue 6) pp:3476
Publication Date(Web):February 13, 2015
DOI:10.1021/acs.analchem.5b00006
A facile approach was developed for direct preparation of organic monoliths via the alkaline-catalyzed thiol-epoxy click polymerization. Two organic monoliths were prepared by using tetraphenylolethane glycidyl ether as a multiepoxy monomer, and trimethylolpropane tris(3-mercaptopropionate) and pentaerythritol tetrakis(3-mercaptopropionate) as the multithiol monomer, respectively, in the presence of a ternary porogenic system (DMSO/PEG200/H2O). The obtained organic monoliths showed high thermal, mechanical and chemical stabilites. Benefiting from the step-growth polymerization process, two organic monoliths possessed well-defined 3D framework microstructure, and exhibited high permeabilities and column efficiencies in capillary liquid chromatography. A series of neutral, basic and acidic small molecules were used to comprehensively evaluate the separation abilities of these monoliths, and satisfactory chromatographic performance with column efficiencies ranged from 35 500 to 132 200 N/m was achieved, demonstrating good separation abilities of these organic monoliths prepared via thiol-epoxy click polymerization approach. Besides, multiple retention mechanisms, including hydrophobic, hydrophilic and π–π conjugate interactions were observed during the separation of analytes on these monoliths, which would make them promising for more extensive applications in capillary liquid chromatography.
Co-reporter:Haiyang Zhang, Junjie Ou, Yinmao Wei, Hongwei Wang, Zhongshan Liu, Lianfang Chen, Hanfa Zou
Analytica Chimica Acta 2015 Volume 883() pp:90-98
Publication Date(Web):9 July 2015
DOI:10.1016/j.aca.2015.04.001
•A crosslinker with multiple acrylate groups was first used for preparing polymeric monoliths.•The poly(LMA-co-DPEPA) monoliths exhibited the column efficiencies of 111,000–165,000 N m−1 for alkylbenzenes in cLC.•Highly crosslinked monoliths also exhibited retention-independent efficient chromatographic performance.Low column efficiency for small molecules in reversed-phase chromatography is a major problem commonly encountered in polymer-based monoliths. Herein, a novel highly crosslinked porous polymeric monolith was in situ prepared by using a multi-acrylate monomer, dipentaerythritol penta-/hexa-acrylate (DPEPA), as crosslinker, which copolymerized with lauryl methacrylate (LMA) as functional monomer in a UV-transparent fused-silica capillary via photo-initiated free-radical polymerization within 5 min. The mechanical stability and permeability of the resulting poly(LMA-co-DPEPA) monolith were characterized in detail. One series of highly crosslinked poly(LMA-co-DPEPA) columns were prepared with relatively higher content of crosslinker (63.3%) in the precursor. Although they exhibited lower permeability, high column efficiency for alkylbenzenes was acquired in cLC, and the minimum plate height (column B) was in the range of 6.04–9.00 μm, corresponding to 111,000–165,000 N m−1. Meanwhile, another series of poly(LMA-co-DPEPA) columns prepared with relatively lower content of crosslinker (52.7%) in the precursor exhibited higher permeability, but the minimum plate height (column E) was relatively low in the range of 10.75–20.04 μm for alkylbenzenes, corresponding to 50,000–93,000 N m−1. Compared with common poly(LMA-co-EDMA) columns previously reported, the highly crosslinked poly(LMA-co-DPEPA) columns using a multi-acrylate monomer as crosslinker possessed remarkably high column efficiency for small molecules in cLC. By plotting of plate height (H) of alkylbenzenes versus the linear velocity (u) of mobile phase, the results revealed a retention-independent efficient performance of small molecules in the isocratic elution, indicating that the use of multi-functional crosslinker possibly prevents the generation of gel-like micropores in the poly(LMA-co-DPEPA) monolith, reducing the mass transfer resistance (C-term).
Co-reporter:Lianfang Chen, Junjie Ou, Zhongshan Liu, Hui Lin, Hongwei Wang, Jing Dong, Hanfa Zou
Journal of Chromatography A 2015 Volume 1394() pp:103-110
Publication Date(Web):15 May 2015
DOI:10.1016/j.chroma.2015.03.054
•A fast approach was successfully developed to prepare an organic monolith via thiol-ene click polymerization.•Effects of the compositions of prepolymerization mixture and reaction time were investigated.•The obtained organic monolith exhibited a very high column efficiency of ∼133,000 plates per meter.A novel organic monolith was firstly prepared in a UV-transparent fused-silica capillary by a single-step approach via photo-initiated thiol-ene click polymerization reaction of 1,2,4-trivinylcyclohexane (TVCH) and pentaerythriol tetra(3-mercaptopropionate) (4SH) within 10 min. The effects of both composition of prepolymerization solution and polymerization time on the morphology and permeability of monolithic column were investigated in detail. Then, the optimal condition was acquired to fabricate a homogeneous and permeable organic monolith. The chemical groups of the monolithic column were confirmed by Fourier transform infrared spectroscopy (FT-IR). The SEM graphs showed the organic monolith possessed a uniform porous structure, which promotes the highest column efficiency of ∼133,000 plates per meter for alkylbenzenes at the linear velocity of 0.65 mm/s in reversed-phase liquid chromatography. Finally, the organic monolithic column was further applied for separation of basic compounds, pesticides and EPA610, indicating satisfactory separation ability.
Co-reporter:Hui Lin, Junjie Ou, Zhongshan Liu, Hongwei Wang, Jing Dong, Hanfa Zou
Journal of Chromatography A 2015 Volume 1379() pp:34-42
Publication Date(Web):30 January 2015
DOI:10.1016/j.chroma.2014.12.031
•Three hybrid monoliths were facilely prepared via thiol-methacrylate Michael addition click reaction.•The obtained monoliths possess high thermal and chemical stabilities.•The highest column efficiency of ca. 190,000 N/m was achieved.•The obtained monoliths exhibited high separation capabilities for various simple and complicated samples.A facile approach based on thiol-methacrylate Michael addition click reaction was developed for construction of porous hybrid monolithic materials. Three hybrid monoliths were prepared via thiol-methacrylate click polymerization by using methacrylate-polyhedral oligomeric silsesquioxane (POSS) (cage mixture, n = 8, 10, 12, POSS-MA) and three multi-thiol crosslinkers, 1,6-hexanedithiol (HDT), trimethylolpropane tris(3-mercaptopropionate) (TPTM) and pentaerythritol tetrakis(3-mercaptopropionate) (PTM), respectively, in the presence of porogenic solvents (n-propanol and PEG 200) and a catalyst (dimethylphenylphosphine, DMPP). The obtained monoliths possessed high thermal and chemical stabilities. Besides, they all exhibited high column efficiencies and excellent separation abilities in capillary liquid chromatography (cLC). The highest column efficiency could reach ca. 195,000 N/m for butylbenzene on the monolith prepared with POSS-MA and TPTM (monolith POSS-TPTM) in reversed-phase (RP) mode at 0.64 mm/s. Good chromatographic performance were all achieved in the separations of polycyclic aromatic hydrocarbons (PAHs), phenols, anilines, EPA 610 as well as bovine serum albumin (BSA) digest. The high column efficiencies in the range of 51,400–117,000 N/m (achieved on the monolith POSS-PTM in RP mode) convincingly demonstrated the high separation abilities of these thiol-methacrylate based hybrid monoliths. All the results demonstrated the feasibility of the phosphines catalyzed thiol-methacrylate Michael addition click reaction in fabrication of monolithic columns with high efficiency for cLC applications.
Co-reporter:Hui Lin, Lianfang Chen, Junjie Ou, Zhongshan Liu, Hongwei Wang, Jing Dong, Hanfa Zou
Journal of Chromatography A 2015 Volume 1416() pp:74-82
Publication Date(Web):16 October 2015
DOI:10.1016/j.chroma.2015.09.011
•Two hybrid monoliths were facilely prepared via thiol–epoxy click polymerization reaction.•The obtained monoliths possess high thermal and chemical stabilities.•The highest column efficiency of ca. 182,700 N/m was achieved.•The obtained monoliths exhibited high separation capabilities for various simple and complicated samples.Two kinds of hybrid monoliths were first prepared via thiol–epoxy click polymerization using a multi-epoxy monomer, octaglycidyldimethylsilyl POSS (POSS-epoxy), and two multi-thiols, trimethylolpropanetris(3-mercaptopropionate) (TPTM) and pentaerythritoltetrakis(3-mercaptopropionate) (PTM), respectively, as the precursors. The resulting two hybrid monoliths (assigned as POSS-epoxy–TPTM and POSS-epoxy–PTM) not only possessed high thermal, mechanical and chemical stabilities, but also exhibited well-controlled 3D skeletal microstructure and high efficiency in capillary liquid chromatography (cLC) separation of small molecules. The highest column efficiency reached 182,700 N/m (for butylbenzene) on the monolith POSS-epoxy–PTM at the velocity of 0.75 mm/s. Furthermore, the hybrid monolith POSS-epoxy–PTM was successfully applied for cLC separations of various samples, not only standard compounds such as alkylbenzenes, PAHs, phenols and dipeptides, as well as intact proteins, but also complex samples of EPA 610 and BSA digest.
Co-reporter:Jinan Li, Fangjun Wang, Hao Wan, Jing Liu, Zheyi Liu, Kai Cheng, Hanfa Zou
Journal of Chromatography A 2015 Volume 1425() pp:213-220
Publication Date(Web):18 December 2015
DOI:10.1016/j.chroma.2015.11.044
•A novel adsorbent coated with branched PEI and maltose was prepared.•The adsorbent exhibited high hydrophilicity and selectivity.•The adsorbent exhibited high N-glycopeptide enrichment capacity (>150 mg/g, IgG/MNPs).•The adsorbent exhibited high N-glycopeptide enrichment recovery (85%).•1567 N-glycopeptides were enriched from the complex protein sample.Hydrophilic interaction chromatography (HILIC) adsorbents have drawn increasing attention in recent years due to their high efficiency in N-glycopeptides enrichment. The hydrophilicity and binding capacity of HILIC adsorbents are crucial to the enrichment efficiency and mass spectrometry (MS) detection sensitivity of N-glycopeptides. Herein, magnetic nanoparticles coated with maltose-functionalized polyethyleneimine (Fe3O4-PEI-Maltose MNPs) were prepared by one-pot solvothermal reaction coupled with “click chemistry” and utilized for N-glycopeptides enrichment. Owing to the presence of hydrophilic and branched polyethyleneimine, the amount of immobilized disaccharide units was improved about four times. The N-glycopeptides capturing capacity was about 150 mg/g (IgG/MNPs) and the MS detection limitation as low as 0.5 fmol for IgG and 85% average enrichment recovery were feasibly achieved by using this hybrid magnetic adsorbent. Finally, 1237 unique N-glycosylation sites and 1567 unique N-glycopeptides from 684 N-glycoproteins were reliably characterized from 60 μg protein sample extracted from mouse liver. Therefore, this maltose-functionalized polyethyleneimine coated adsorbent can play a promising role in highly efficient N-glycopeptides enrichment for glycoproteomic analyses of complex protein samples.
Co-reporter:Zhang Zhang, Deguang Sun, Yuting Cong, Jiawei Mao, Junfeng Huang, Hongqiang Qin, Jing Liu, Guang Huang, Liming Wang, Mingliang Ye, and Hanfa Zou
Journal of Proteome Research 2015 Volume 14(Issue 9) pp:3892-3899
Publication Date(Web):August 10, 2015
DOI:10.1021/acs.jproteome.5b00306
An amine chemistry method was developed for the extraction of N-glycopeptides using amine-functionalized beads for glycoproteomics analysis. Two reductive amination reactions between primary amine and aldehyde were employed in this approach. The first one was to block the primary amines in the peptides by addition of formaldehyde and sodium cyanoborohydride into the peptide sample, and the second one was to couple the glycopeptides onto solid phase beads by incubating the glycopeptides containing aldehyde groups (oxidized by periodate) with the amine-functionalized beads in the presence of sodium cyanoborohydride. It was demonstrated that the blocking of primary amines in the peptides by the first reductive amination reaction prior to the periodate oxidation made the amine chemistry method very efficient and sensitive. This new method was validated by analysis of glycoprotein standards as well as proteome samples. It was found that this new method led to significant increase in the identification of N-glycosites compared with the conventional hydrazide chemistry method.
Co-reporter:Mingming Dong; Yangyang Bian; Jing Dong; Keyun Wang; Zheyi Liu; Hongqiang Qin; Mingliang Ye
Journal of Proteome Research 2015 Volume 14(Issue 12) pp:5341-5347
Publication Date(Web):November 10, 2015
DOI:10.1021/acs.jproteome.5b00830
Among the natural amino acids, cysteine is unique since it can form a disulfide bond through oxidation and reduction of sulfhydryl and thus plays a pervasive role in modulation of proteins activities and structures. Crosstalk between phosphorylation and other post-translational modifications has become a recurrent theme in cell signaling regulation. However, the crosstalk between the phosphorylation and the formation and reductive cleavage of disulfide bond has not been investigated so far. To facilitate the study of this crosstalk, it is important to explore the subset of phosphoproteome where phosphorylations are occurred near to cysteine in the protein sequences. In this study, we developed a straightforward sequential enrichment method by combining the thiol affinity chromatography with the immobilized titanium ion affinity chromatography to selectively enrich cysteine-containing phosphopeptides. The high specificity and high sensitivity of this method were demonstrated by analyzing the samples of Jurkat cells. This “divide and conquer” strategy by specific analysis of a subphosphoproteome enables identification of more low abundant phosphosites than the conventional global phosphoproteome approach. Interestingly, amino acid residues surrounding the identified phosphosites were enriched with buried residues (L, V, A, C) while depleted with exposed residues (D, E, R, K). Also, the phosphosites identified by this approach showed a dramatic decrease in locating in disorder regions compared to that identified by conventional global phosphoproteome. Further analysis showed that more proline directed kinases and fewer acidophilic kinases were responsible for the phosphorylation sites of this subphosphoproteome.
Co-reporter:Keyun Wang, Yongjin J. Zhou, Hongwei Liu, Kai Cheng, Jiawei Mao, Fangjun Wang, Wujun Liu, Mingliang Ye, Zongbao K. Zhao, Hanfa Zou
Journal of Proteomics 2015 Volume 114() pp:226-233
Publication Date(Web):30 January 2015
DOI:10.1016/j.jprot.2014.07.032
•The methylation by isotope-labeled SAM (MILS) strategy developed•A comprehensive yeast methylproteome presented•The amino acid residue preference of protein methylation in yeast establishedProtein methylation catalyzed by SAM-dependent methyltransferase represents a major PTM involved in many important biological processes. Because methylation can occur on nitrogen, oxygen and sulfur centers and multiple methylation states exist on the nitrogen centers, methylproteome remains poorly documented. Here we present the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast Saccharomyces cerevisiae based on the online multidimensional μHPLC/MS/MS technology. We identified 43 methylated proteins, containing 68 methylation events associated with 64 methylation sites. More than 90% of these methylation events were previously unannotated in Uniprot database. Our results indicated, 1) over 2.6% of identified S. cerevisiae proteins are methylated, 2) the amino acid residue preference of protein methylation follows the order Lys ≫ Arg > Asp > Asn ≈ Gln ≈ His > Glu > Cys, and 3) the methylation state on nitrogen center is largely exclusive. As our dataset covers various types of methylation centers, it provides rich information about yeast methylproteome and should significantly contribute to the field of protein methylation.Biological significanceIn this paper, we presented the methylation by isotope labeled SAM (MILS) strategy for a highly-confident analysis of the methylproteome of the yeast S. cerevisiae and collected a comprehensive list of proteins methylated on a set of distinct residues (K, R, N, E, D, Q, H, C). Our study provided useful information about the amino acid residue preference and methylation state distributions on nitrogen centers of protein methylation in S. cerevisiae.
Co-reporter:Baofeng Zhao, Bo Xu, Wenquan Hu, Chunxia Song, Fangjun Wang, Zhong Liu, Mingliang Ye, Hanfa Zou, Qing R. Miao
Journal of Proteomics 2015 Volume 112() pp:38-52
Publication Date(Web):1 January 2015
DOI:10.1016/j.jprot.2014.08.007
•Pseudo triplex labeling approach is suitable for analysis of biological samples.•NgBR deficiency diminishes the expression of mesenchymal cell markers.•NgBR deficiency results in the MET of MDA-MB-231 breast tumor cells.•NgBR deficiency prevents the TGF-β induced EMT of MCF-7 breast tumor cells.•NgBR may play an important role in switching decision between EMT and MET.Nogo-B receptor (NgBR) is a type I receptor and specifically binds to ligand Nogo-B. Our previous work has shown that NgBR is highly expressed in human breast invasive ductal carcinoma. Here, comprehensive proteome quantification was performed to examine the alteration of protein expression profile in MDA-MB-231 breast tumor cells after knocking down NgBR using lentivirus-mediated shRNA approach. Among a total of 1771 proteins feasibly quantified, 994 proteins were quantified in two biological replicates with RSD < 50%. There are 122 proteins significantly down-regulated in NgBR knockdown MDA-MB-231 breast tumor cells, such as vimentin and S100A4, well-known markers for mesenchymal cells, and CD44, a stemness indicator. The decrease of vimentin, S100A4 and CD44 protein expression levels was further confirmed by Western blot analysis. MDA-MB-231 cells are typical breast invasive ductal carcinoma cells showing mesenchymal phenotype. Cell morphology analysis demonstrates NgBR knockdown in MDA-MB-231 cells results in reversibility of epithelial–mesenchymal transition (EMT), which is one of the major mechanisms involved in breast cancer metastasis. Furthermore, we demonstrated that NgBR knockdown in MCF-7 cells significantly prevented the TGF-β-induced EMT process as determined by the morphology change, and staining of E-cadherin intercellular junction as well as the decreased expression of vimentin.Biological significanceOur previous publication showed that NgBR is highly expressed in human breast invasive ductal carcinoma. However, the roles of NgBR and NgBR-mediated signaling pathway in breast tumor cells are still unclear. Here, we not only demonstrated that the quantitative proteomics analysis is a powerful tool to investigate the global biological function of NgBR, but also revealed that NgBR is involved in the transition of breast epithelial cells to mesenchymal stem cells, which is one of the major mechanisms involved in breast cancer metastasis. These findings provide new insights for understanding the roles of NgBR in regulating breast epithelial cell transform during the pathogenesis of breast cancer.
Co-reporter:Fang-Jie LIU, Ming-Liang YE, Yan-Bo PAN, Han-Fa ZOU
Chinese Journal of Analytical Chemistry 2015 Volume 43(Issue 10) pp:1452-1458
Publication Date(Web):October 2015
DOI:10.1016/S1872-2040(15)60864-7
Polyacrylamide gel electrophoresis (PAGE) is a powerful protein separation technology. Combined with mass spectrometry, it could identify thousands of proteins in proteomics analysis. However, the time-consuming procedure restricts its broad applications in proteomics study. In this work, it was found that high concentration of trypsin did not compromise subsequent phosphopeptide enrichment after in-gel digestion, but could promote the in-gel digestion. Hence, a new, fast and robust digestion method was established. Firstly, 50 μg of HeLa cell protein was separated into 5 fractions by SDS-PAGE, and then the proteins were digested with high concentration trypsin for 30 min. Finally, the phosphopeptides were enriched by Ti-IMAC Tip. After liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, about 2000 phosphorylation sites were identified in the experimental group, while less than 1500 phosphorylation sites were identified in control group. With the aid of high concentration of trypsin, in-gel digestion could be completed within only 30 min, and more phosphorylation site identifications and lower percentage of missed cleavages were acquired than those in control experiments. The experiment results demonstrated that high concentration of trypsin could not only accelerate the in-gel digestion, but also improve the phosphoproteome coverage.Figure A was the process of polyacrylamide gel electrophoresis. The proteins were separated by their molecular weights. B was the process of in gel digestion assisted by high concentration of trypsin. C were the acquired peptides extracted from the gel slices. D were the purified phosphopeptides through Ti-IMAC enrichment.
Co-reporter:Hao Wan, Yi Zhang, Zheyi Liu, Guiju Xu, Guang Huang, Yongsheng Ji, Zhichao Xiong, Quanqing Zhang, Jing Dong, Weibing Zhang and Hanfa Zou
Nanoscale 2014 vol. 6(Issue 15) pp:8743-8753
Publication Date(Web):09 May 2014
DOI:10.1039/C4NR01044B
Remote-controlled nanocarriers for drug delivery are of great promise to provide timely, sensitive and spatiotemporally selective treatments for cancer therapy. Due to convenient and precise manipulation, deep penetration through tissues and excellent biocompatibility, near-infrared (NIR) irradiation is a preferred external stimulus for triggering the release of loaded drugs. In this work, for spatiotemporally controlled chemo-photothermal synergistic cancer therapy, a NIR responsive nanocarrier was fabricated using reduced graphene oxide nanosheets (rNGO) decorated with mesoporous silica shell and the subsequent functionalization of the thermoresponsive polymer brushes (pNIPAM-co-pAAm) at the outlet of the silica pore channels. rNGO, which combined with the mesoporous silica shell provide a high loading capacity for anticancer drugs (doxorubicin, DOX), was assigned to sense NIR irradiation for the manipulation of pNIPAM-co-pAAm valve to control the diffusion of loaded DOX. Under NIR irradiation, rNGO would generate heat, which could not only elevate the surrounding temperature over the low critical solution temperature (LCST) of pNIPAM-co-pAAm to open the thermoresponsive polymer valve and promote the diffusion of DOX, but also kill the cancer cells through the hypothermia effect. By manipulating NIR irradiation, the nanocarrier exhibited efficiently controlled release of loaded DOX both in the buffer and in living HeLa cells (the model cancer cells), providing powerful and site-targeted treatments, which can be attributed to synergistic effects of chemo-photothermal therapy. To sum up, this novel nanocarrier is an excellent drug delivery platform in remote-controlled chemo-photothermal synergistic cancer therapy via NIR irradiation.
Co-reporter:Zhichao Xiong, Lingyi Zhang, Chunli Fang, Quanqing Zhang, Yongsheng Ji, Zhang Zhang, Weibing Zhang and Hanfa Zou
Journal of Materials Chemistry A 2014 vol. 2(Issue 28) pp:4473-4480
Publication Date(Web):08 May 2014
DOI:10.1039/C4TB00479E
Highly selective and efficient enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel immobilized metal affinity chromatography (IMAC) material has been synthesized to improve the enrichment specificity and sensitivity for phosphopeptides by introducing a titanium phosphate moiety on a multilayer polysaccharide (hyaluronate (HA) and chitosan (CS)) coated Fe3O4@SiO2 nanoparticle (denoted as Fe3O4@SiO2@(HA/CS)10–Ti4+ IMAC). The thicker multilayer polysaccharide endows excellent hydrophilic properties and a higher binding capacity of the titanium ion to the IMAC material. Due to the combination of uniform magnetic properties, highly hydrophilic properties and enhanced binding capacity of the titanium ion, the Fe3O4@SiO2@(HA/CS)10–Ti4+ nanoparticle possesses many merits, such as high selectivity for phosphopeptides (phosphopeptides/non-phosphopeptides at a molar ratio of 1:2000), extreme detection sensitivity (0.5 fmol), large binding capacity (100 mg g−1), high enrichment recovery (85.45%) and rapid magnetic separation (within 10 s). Moreover, the as-prepared IMAC nanoparticle provides effective enrichment of phosphopeptides from real samples (human serum and nonfat milk), showing great potential as a tool for the detection and identification of low-abundance phosphopeptides in biological samples.
Co-reporter:Zhichao Xiong, Yajing Chen, Lingyi Zhang, Jun Ren, Quanqing Zhang, Mingliang Ye, Weibing Zhang, and Hanfa Zou
ACS Applied Materials & Interfaces 2014 Volume 6(Issue 24) pp:22743
Publication Date(Web):December 3, 2014
DOI:10.1021/am506882b
The highly selective and efficient capture of heterogeneous types of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis, but it still remains a challenge since the lack of affinity material with large binding capacity and controllable specificity. Here, a new affinity material was prepared to improve the enrichment capacity and endue the tunable specificity by introducing guanidyl onto poly(glycidyl methacrylate) (PGMA) modified Fe3O4 microsphere (denoted as Fe3O4@PGMA-Guanidyl). The thick polymer shell endows the composite microsphere with large amount of guanidyl and is beneficial to enhancing the affinity interaction between phosphopeptides and the material. Interestingly, the Fe3O4@PGMA-Guanidyl possesses tunable enriching ability for global phosphopeptides or only multiphosphopeptides through simple regulation of buffer composition. The composite has large enrichment capacity (200 mg g–1), extremely high detection sensitivity (0.5 fmol), high enrichment recovery (91.30%), great specificity, and rapid magnetic separation. Moreover, the result of the application to capture of phosphopeptides from tryptic digest of nonfat milk has demonstrated the great potential of Fe3O4@PGMA-Guanidyl in detection and identification of low-abundance phosphopeptides of interest in biological sample.Keywords: guanidyl; magnetic microspheres; mass spectrum; phosphorylated peptides; tunable enrichment ability
Co-reporter:Zhongshan Liu, Junjie Ou, Hui Lin, Zheyi Liu, Hongwei Wang, Jing Dong and Hanfa Zou
Chemical Communications 2014 vol. 50(Issue 66) pp:9288-9290
Publication Date(Web):12 Jun 2014
DOI:10.1039/C4CC03451A
Hybrid monoliths with a macroporous structure were prepared within a few minutes via a photoinduced thiol–ene polymerization reaction, the surfaces of which showed hydrophobic character. The monolithic column demonstrated good separation performance towards alkylbenzenes, peptides, proteins and BSA tryptic digest in cLC.
Co-reporter:Zhenzhen Deng, Mingliang Ye, Yangyang Bian, Zheyi Liu, Fangjie Liu, Chunli Wang, Hongqiang Qin and Hanfa Zou
Chemical Communications 2014 vol. 50(Issue 90) pp:13960-13962
Publication Date(Web):11 Sep 2014
DOI:10.1039/C4CC04906C
A simple, cost-effective and high throughput method was developed for multiplexed kinase activity assay based on the multiplex isotope labeling of designed substrate peptides. This strategy was successfully applied to monitor the time-dependent consumption of substrates and generation of products in the single and multiple substrate systems.
Co-reporter:Yue Wu, Fangjun Wang, Zheyi Liu, Hongqiang Qin, Chunxia Song, Junfeng Huang, Yangyang Bian, Xiaoluan Wei, Jing Dong and Hanfa Zou
Chemical Communications 2014 vol. 50(Issue 14) pp:1708-1710
Publication Date(Web):09 Dec 2013
DOI:10.1039/C3CC47998F
Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.
Co-reporter:Zhongshan Liu, Junjie Ou, Hui Lin, Hongwei Wang, Zheyi Liu, Jing Dong, and Hanfa Zou
Analytical Chemistry 2014 Volume 86(Issue 24) pp:12334
Publication Date(Web):November 18, 2014
DOI:10.1021/ac503626v
Two monolithic polymer columns were directly prepared in the UV-transparent fused-silica capillaries via photoinitiated thiol-yne click polymerization of 1,7-octadiyne (ODY) with a dithiol (1,6-hexanedithiol, 2SH) or a tetrathiol (pentaerythriol tetrakis(3-mercaptopropionate), 4SH) within 15 min. The rapid polymerization provided a time-saving approach to optimize preparation conditions. Then, two porogenic systems of diethylene glycol diethyl ether (DEGDE)/tetrahydrofuran (THF) and DEGDE/poly(ethylene glycol) (PEG, Mn = 200) were found to effectively control the porous structure of two kinds of polymeric monoliths (O2SH and O4SH), respectively. The almost disappearance of thiol and alkynyl vibrations (2560 and 2115 cm–1, respectively) in infrared spectra and Raman spectra indicated a high conversion of the thiol-yne polymerization reaction. The thiol-yne polymerization was further proved by analyzing the energy-dispersive X-ray spectrum (EDS), MALDI-TOF mass spectrum, and elemental data. Scanning electron microscopy (SEM) images showed the monolithic polymer columns with homogeneous porous structure and macropore size of 0.5–1.0 μm, which facilitated the minimum plate heights of 10.0–12.0 μm for alkylbenzenes in reversed-phase liquid chromatography (RPLC). The low values of the A and C terms (<1.0 μm and <15.5 ms, respectively) in the van Deemter equation were similar to those obtained by some monolithic silica columns. The BSA tryptic digest was also separated on the monolithic polymer column by cLC-MS/MS. The result with 85% protein coverage was better than those given by some hybrid monolithic columns. The monolithic polymer columns were further applied for separation of phenols, natural products, and standard proteins and demonstrated satisfactory separation ability.
Co-reporter:Zhang Zhang, Zhen Sun, Jun Zhu, Jing Liu, Guang Huang, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2014 Volume 86(Issue 19) pp:9830
Publication Date(Web):September 10, 2014
DOI:10.1021/ac5024638
Sialylated glycoproteins, which play important roles in tumor progression, have been extensively analyzed for the discovery of potential biomarkers for cancer diagnosis and prognosis. The site-specific N-sialoglycan occupancy rates of glycoproteins reflect the activities of glycosyltransferases and glycosidases in vivo and could be novel disease biomarkers. However, a high-throughput method to determine the N-sialoglycan occupancy rates is not available. On the basis of the fact that dihydroxy of sialic acid of glycan chains in glycoproteins can be specifically oxidized to aldehyde in mild periodate concentration while all types of glycan chains can be oxidized in high periodate concentration, we developed a modified protein-level hydrazide chemistry method for the determination of the N-sialoglycan occupancy rates. This method was first applied to determine the N-sialoglycan occupancy rates of two glycosites on human transferrin. These two sites were found to be fully sialylated and the N-sialoglycan occupancy rates were found to under significant decrease after the neuraminidase treatment. This method was then applied to analyze N-sialoglycan occupancy rates in proteome samples. We determined 496 and 632 site-specific N-sialoglycan occupancy rates on 334 and 394 proteins from hepatocellular carcinoma (HCC) and normal human liver tissues, respectively. By comparing the N-sialoglycan occupancy rates between the above two samples, we determined 76 N-sialoglycosites with more than a 2-fold change. This method was demonstrated to be an effective and high-throughput method for the analysis of the N-sialoglycan occupancy rates.
Co-reporter:Yanbo Pan, Mingliang Ye, Hao Zheng, Kai Cheng, Zhen Sun, Fangjie Liu, Jing Liu, Keyun Wang, Hongqiang Qin, and Hanfa Zou
Analytical Chemistry 2014 Volume 86(Issue 2) pp:1170
Publication Date(Web):December 19, 2013
DOI:10.1021/ac403060d
An enzymatic approach to label peptide N-termini with isotope-coded affinity tags is presented. This method exploits the high activity of trypsin for peptide synthesis in organic solvents. A cosubstrate containing a stable isotope-coded Arg residue and a biotin tag was synthesized. When the cosubstrate was incubated with tryptic peptides and trypsin in ethanol solution, the stable isotope-coded affinity tag was specifically coupled onto the N-termini of peptides via the formation of new peptide bonds. The labeled peptides were specifically enriched by avidin affinity chromatography and then were submitted to liquid chromatography–tandem mass spectrometry (LC/MS/MS) for quantification. This enrichment step effectively reduced the interference by unlabeled peptides. The excellent performance of this approach was demonstrated by labeling standard peptides as well as a mouse liver digest. In addition to one amino acid residue, a few dipeptide tags were also introduced to the N-termini of peptides successfully by this enzymatic approach. It was found that the identifications for samples labeled with these tags were highly complementary. Coupling a short sequence tag onto peptides could be an effective approach to improve the coverage for proteome analysis.
Co-reporter:Fangjie Liu, Mingliang Ye, Yanbo Pan, Yi Zhang, Yangyang Bian, Zhen Sun, Jun Zhu, Kai Cheng, and Hanfa Zou
Analytical Chemistry 2014 Volume 86(Issue 14) pp:6786
Publication Date(Web):June 23, 2014
DOI:10.1021/ac5002146
Conventional sample preparation protocols for phosphoproteome analysis require multiple time-consuming and labor-intensive steps, including cell lysis, protein extraction, protein digestion, and phosphopeptide enrichment. In this study, we found that the presence of a large amount of trypsin in the sample did not interfere with phosphopeptide enrichment and subsequent LC-MS/MS analysis. Taking advantage of fast digestion achieved with high trypsin-to-protein ratio, we developed a novel concurrent lysis-digestion method for phosphoproteome analysis. In this method, the harvested cells were first placed in a lysis buffer containing a huge amount of trypsin. After ultrasonication, the cells were lysed and the proteins were efficiently digested into peptides within one step. Thereafter, tryptic digest was subjected to phosphopeptide enrichment, in which unphosphorylated peptides, trypsin, and other components incompatible with LC-MS/MS analysis were removed. Compared with conventional methods, better phosphoproteome coverage was achieved in this new one-step method. Because protein solubilization and cell lysis were facilitated by fast protein digestion, the complete transformation of cell pellets into the peptide mixture could be finished within 25 min, while it would take at least 16 h for conventional methods. Hence, our method, which integrated cell lysis, protein extraction, and protein digestion into one step, is rapid and convenient. It is expected to have broad applications in phosphoproteomics analysis.
Co-reporter:Chunli Fang, Zhichao Xiong, Hongqiang Qin, Guang Huang, Jing Liu, Mingliang Ye, Shun Feng, Hanfa Zou
Analytica Chimica Acta 2014 Volume 841() pp:99-105
Publication Date(Web):2 September 2014
DOI:10.1016/j.aca.2014.05.037
•Chitosan coated MCNC materials were prepared by a simple one-pot method.•The Fe3O4@CS MCNC materials showed super hydrophilicity.•The material had high selectivity and efficiency for enrichment of glycopeptides.Selective enrichment of glycopeptides prior to the mass spectrometry (MS) analysis is essential due to ion suppression effect during ionization caused by the co-presence of non-glycosylated peptides. Among the enrichment approaches, hydrophilic interaction liquid chromatography (HILIC) based on magnetic separation has become a popular method in recent years. As the conventional synthesis procedures of these materials are tedious and time-consuming with at least four steps. Herein, magnetic colloidal nanocrystal clusters coated with chitosan (Fe3O4@CS MCNCs) have been successfully prepared by a simple one-pot method. The resulting Fe3O4@CS MCNCs demonstrated an excellent ability for glycopeptide enrichment with high selectivity, low detection limit and high binding capacity. Furthermore, in the analysis of real complicated biological sample, 283 unique N-glycosylation sites corresponding to 175 glycosylated proteins were identified in three replicate analyses of 45 μg protein sample extracted from HeLa cells, indicating the great potential in detection and identification of low abundant glycopeptides in glycoproteome analysis.
Co-reporter:Guang Huang, Zhichao Xiong, Hongqiang Qin, Jun Zhu, Zhen Sun, Yi Zhang, Xiaojun Peng, Junjie ou, Hanfa Zou
Analytica Chimica Acta 2014 Volume 809() pp:61-68
Publication Date(Web):27 January 2014
DOI:10.1016/j.aca.2013.11.049
•Silica nanoparticles modified with uniform PMSA brushes (SiO2-RAFT@PMSA) via RAFT technique were successfully synthesized.•The SiO2-RAFT@PMSA nanoparticles were successfully applied for glycopeptide enrichment.•SiO2-RAFT@PMSA nanoparticles performed better glycopeptide enrichment than monolayer materials & PMSA brushes nanoparticles.Zwitterionic hydrophilic interaction chromatography (ZIC-HILIC) materials have been increasingly attractive in glycopeptide enrichment. However, the traditional ZIC-HILIC materials are modified with monolayer zwitterionic molecules on the surface, therefore, the hydrophilicity, detection sensitivity and loading capacity are limited. In this work, we synthesized novel silica nanoparticles with uniform poly(2-(methacryloyloxy)ethyl)dimethyl-(3-sul-fopropyl)ammonium hydroxide (PMSA) brushes grafted onto the surface via reversible addition-fragmentation chain transfer (RAFT) polymerization (denoted as SiO2-RAFT@PMSA). The resulting SiO2-RAFT@PMSA nanoparticles demonstrated low detection limit (10 fmol) and high recovery yield (over 88%) for glycopeptide enrichment from tryptic digest of human IgG. The SiO2-RAFT@PMSA nanoparticles were further applied for the analysis of mouse liver glycoproteome, a total number of 303 unique N-glycosylation sites corresponding to 185 glycoproteins was reliably profiled in three replicate nano-LC–MS/MS runs. Significantly, more glycopeptides were identified than those of nanoparticles, monolayer MSA molecules modified SiO2@single-MSA and nonuniform multi-layer PMSA brushes coated SiO2@PMSA, as well as commercial ZIC@HILIC beads and Click Maltose beads. The excellent performance of SiO2-RAFT@PMSA nanoparticles results from the non-fouling property, a large quantity of functional molecules and suitable link arms provided by uniform PMSA brushes, as well as efficient interaction between glycopeptides and uniform PMSA brushes. It is concluded that the synthesized SiO2-RAFT@PMSA nanoparticles exhibit great potential in glycoproteome analysis. Moreover, this strategy to modify nanopaticles with uniform polymer brushes via RAFT polymerization can also be explored to design other types of materials for bioseparation application.
Co-reporter:Zhongshan Liu, Junjie Ou, Hui Lin, Hongwei Wang, Jing Dong, Hanfa Zou
Journal of Chromatography A 2014 Volume 1342() pp:70-77
Publication Date(Web):16 May 2014
DOI:10.1016/j.chroma.2014.03.058
•A facile approach was successfully developed to prepare a POSS-based hybrid monolith possessing disulfide bonds.•Functionalization of POSS-based hybrid monolith was performed with methylacrylate via thiol-ene reaction at room temperature.•The column efficiency was obviously enhanced after SMA-modification of hybrid monolithic column.•A 1-m-long column of 75 μm i.d. was prepared and exhibited the highest column efficiency of ∼70,000 plates.A polyhedral oligomeric silsesquioxane (POSS) hybrid monolith was simply prepared by using octaglycidyldimethylsilyl POSS (POSS-epoxy) and cystamine dihydrochloride as monomers via ring-opening polymerization. The effects of composition of prepolymerization solution and polycondensation temperature on the morphology and permeability of monolithic column were investigated in detail. The obtained POSS hybrid monolithic column showed 3D skeleton morphology and exhibited high column efficiency of ∼71,000 plates per meter in reversed-phase mechanism. Owing to this POSS hybrid monolith essentially possessing a great number of disulfide bonds, the monolith surface would expose thiol groups after reduction with dithiothreitol (DTT), which supplied active sites to functionalize with various alkene monomers via thiol-ene click reaction. The results indicated that the reduction with DTT could not destroy the 3D skeleton of hybrid monolith. Both stearyl methylacrylate (SMA) and benzyl methacrylate (BMA) were selected to functionalize the hybrid monolithic columns for reversed-phase liquid chromatography (RPLC), while [2-(methacryloyloxy)ethyl]-dimethyl-(3-sulfopropyl)-ammonium hydroxide (MSA) was used to modify the hybrid monolithic column in hydrophilic interaction chromatography (HILIC). These modified hybrid monolithic columns could be successfully applied for separation of small molecules with high efficiency. It is demonstrated that thiol-ene click reaction supplies a facile way to introduce various functional groups to the hybrid monolith possessing thiol groups. Furthermore, due to good permeability of the resulting hybrid monoliths, we also prepared long hybrid monolithic columns in narrow-bore capillaries. The highest column efficiency reached to ∼70,000 plates using a 1-m-long column of 75 μm i.d. with a peak capacity of 147 for isocratic chromatography, indicating potential application in separation and analysis of complex biosamples.
Co-reporter:Hongwei Wang, Junjie Ou, Hui Lin, Zhongshan Liu, Guang Huang, Jing Dong, Hanfa Zou
Journal of Chromatography A 2014 Volume 1367() pp:131-140
Publication Date(Web):7 November 2014
DOI:10.1016/j.chroma.2014.09.072
•Two hybrid monoliths were prepared by different polymerization techniques.•Two hybrid monoliths were investigated on the morphology and chromatographic assessment.•Both hybrid monoliths showed satisfactory tailorability.Two kinds of hybrid monolithic columns were prepared by using methacrylate epoxy cyclosiloxane (epoxy-MA) as functional monomer, containing three epoxy moieties and one methacrylate group. One column was in situ fabricated by ring-opening polymerization of epoxy-MA and 1,10-diaminodecane (DAD) using a porogenic system consisting of isopropanol (IPA), H2O and ethanol at 65 °C for 12 h. The other was prepared by free radical polymerization of epoxy-MA and ethylene dimethacrylate (EDMA) using 1-propanol and 1,4-butanediol as the porogenic solvents at 60 °C for 12 h. Two hybrid monoliths were investigated on the morphology and chromatographic assessment. Although two kinds of monolithic columns were prepared with epoxy-MA, their morphologies looked rather different. It could be found that the epoxy-MA–DAD monolith possessed higher column efficiencies (25,000–34,000 plates/m) for the separation of alkylbenzenes than the epoxy-MA–EDMA monolith (12,000–13,000 plates/m) in reversed-phase nano-liquid chromatography (nano-LC). Depending on the remaining epoxy or methacrylate groups on the surface of two pristine monoliths, the epoxy-MA–EDMA monolith could be easily modified with 1-octadecylamine (ODA) via ring-opening reaction, while the epoxy-MA–DAD monolith could be modified with stearyl methacrylate (SMA) via free radical reaction. The chromatographic performance for the separation of alkylbenzenes on SMA-modified epoxy-MA–DAD monolith was remarkably improved (42,000-54,000 plates/m) when compared with that on pristine epoxy-MA–DAD monolith, while it was not obviously enhanced on ODA-modified epoxy-MA–EDMA monolith when compared with that on pristine epoxy-MA–EDMA monolith. The enhancement of the column efficiency of epoxy-MA–DAD monolith after modification might be ascribed to the decreased mass-transfer resistence. The two kinds of hybrid monoliths were also applied for separations of six phenols and seven basic compounds in nano-LC.
Co-reporter:Chunxia Song, Fangjun Wang, Kai Cheng, Xiaoluan Wei, Yangyang Bian, Keyun Wang, Yexiong Tan, Hongyang Wang, Mingliang Ye, and Hanfa Zou
Journal of Proteome Research 2014 Volume 13(Issue 1) pp:241-248
Publication Date(Web):2017-2-22
DOI:10.1021/pr400544j
Global quantification of the single amino-acid variations (SAAVs) is essential to investigate the roles of SAAVs in disease progression. However, few efforts have been made on this issue due to the lack of high -throughput approach. Here we presented a strategy by integration of the stable isotope dimethyl labeling with variation-associated database search to globally quantify the SAAVs at the first time. A protein database containing 87 745 amino acid variant sequences and 73 910 UniProtKB/Swiss-Prot canonical protein entries was constructed for database search, and higher energy collisional dissociation combined with collision-induced dissociation fragmentation modes were applied to improve the quantification coverage of SAAVs. Compared with target proteomics in which only a few sites could be quantified, as many as 282 unique SAAVs sites were quantified between hepatocellular carcinoma (HCC) and normal human liver tissues by our strategy. The variation rates in different samples were evaluated, and some interesting SAAVs with significant increase normalized quantification ratios, such as T1406N in CPS1 and S197R in HTATIP2, were observed to highly associate with HCC progression. Therefore, the newly developed strategy enables the large-scale comparative analysis of variations at the protein level and holds a promising future in the research related to variations.
Co-reporter:Jun Zhu, Zhen Sun, Kai Cheng, Rui Chen, Mingliang Ye, Bo Xu, Deguang Sun, Liming Wang, Jing Liu, Fangjun Wang, and Hanfa Zou
Journal of Proteome Research 2014 Volume 13(Issue 3) pp:1713-1721
Publication Date(Web):2017-2-22
DOI:10.1021/pr401200h
Although glycoproteomics is greatly developed in recent years, our knowledge about N-glycoproteome of human tissues is still very limited. In this study, we comprehensively mapped the N-glycosylation sites of human liver by combining click maltose–hydrophilic interaction chromatography (HILIC) and the improved hydrazide chemistry. The specificity could be as high as 90% for hydrazide chemistry and 80% for HILIC. Altogether, we identified 14 480 N-glycopeptides matched with N-!P-[S|T|C] sequence motif from human liver, corresponding to 2210 N-glycoproteins and 4783 N-glycosylation sites. These N-glycoproteins are widely involved into different types of biological processes, such as hepatic stellate cell activation and acute phase response of human liver, which all highly associate with the progression of liver diseases. Moreover, the exact N-glycosylation sites of some key-regulating proteins within different human liver physiological processes were also obtained, such as E-cadherin, transforming growth factor beta receptor and 29 members of G protein coupled receptors family.
Co-reporter:Zhen Sun, Jiaqiang Dong, Song Zhang, Zhengyan Hu, Kai Cheng, Kai Li, Bo Xu, Mingliang Ye, Yongzhan Nie, Daiming Fan, and Hanfa Zou
Journal of Proteome Research 2014 Volume 13(Issue 3) pp:1593-1601
Publication Date(Web):2017-2-22
DOI:10.1021/pr4010822
Chemoresistance remains the most significant obstacle to successful chemotherapy for leukemia, and its exact mechanism is still unknown. In this work, we used the cell-surface capturing method together with quantitative proteomics to investigate differences in the glycoproteomes of adriamycin-sensitive and adriamycin-resistant leukemia cells. Two quantitative methods, isotopic dimethyl labeling and SWATH, were used to quantify glycoproteins, and 35 glycoproteins were quantified by both methods. High correlation was observed between the glycoproteins quantified by the above two methods, and 15 glycoproteins displayed a consistent significant change trend in both sets of quantitative results. These 15 proteins included classical multidrug resistance-related glycoproteins such as ABCB1 as well as a set of novel glycoproteins that have not previously been reported to be associated with chemoresistance in leukemia cells. Further validation with quantitative real-time PCR and Western blotting confirmed the proteomic screening results. Subsequent functional experiments based on RNA interference technology showed that CTSD, FKBP10, and SLC2A1 are novel genes that participate in the acquisition and maintenance of the adriamycin-resistant phenotype in leukemia cells.
Co-reporter:Bo Xu, Fangjun Wang, Chunxia Song, Zhen Sun, Kai Cheng, Yexiong Tan, Hongyang Wang, and Hanfa Zou
Journal of Proteome Research 2014 Volume 13(Issue 8) pp:3645-3654
Publication Date(Web):2017-2-22
DOI:10.1021/pr500200s
Hepatocellular carcinoma is one of the most fatal cancers worldwide. In this study, a reversed-phase–strong cation exchange–reversed-phase three-dimensional liquid chromatography strategy was established and coupled with mass spectrometry to investigate the differential proteome expression of HCC and normal liver tissues. In total, 2759 proteins were reliably quantified, of which, 648 proteins were dysregulated more than 3-fold in HCC liver tissues. Some important proteins that relate to HCC pathology were significantly dysregulated, such as NAT2 and AKR1B10. Furthermore, 2307 phosphorylation sites from 1264 phosphoproteins were obtained in our previous phosphoproteome quantification, and the nonphosphorylated counterparts of 445 phosphoproteins with 983 phosphorylation sites were reliably quantified in this work. It was observed that 337 (34%) phosphorylation sites exhibit significantly different expression trends from that of their corresponding nonphosphoproteins. Some novel phosphorylation sites with important biological functions in the progression of HCC were reliably quantified, such as the significant downregulation of pT185 for ERK2 and pY204 for ERK1.
Co-reporter:Zhengyan Hu, Liang Zhao, Hongyan Zhang, Yi Zhang, Ren’an Wu, Hanfa Zou
Journal of Chromatography A 2014 Volume 1334() pp:55-63
Publication Date(Web):21 March 2014
DOI:10.1016/j.chroma.2014.01.077
•On-bead digestion of protein corona by immobilized trypsin is of great efficiency.•Self-digestion of trypsin has been reduced significantly by using the immobilized trypsin.•The digestion of protein corona with immobilized trypsin demonstrates the improved reproducibility than the conventional free trypsin.Proteins interacting with nanoparticles would form the protein coronas on the surface of nanoparticles in biological systems, which would critically impact the biological identities of nanoparticles and/or result in the physiological and pathological consequences. The enzymatic digestion of protein corona was the primary step to achieve the identification of protein components of the protein corona for the bottom-up proteomic approaches. In this study, the investigation on the tryptic digestion of protein corona by the immobilized trypsin on a magnetic nanoparticle was carried out for the first time. As a comparison with the usual overnight long-time digestion and the severe self-digestion of free trypsin, the on-bead digestion of protein corona by the immobilized trypsin could be accomplished within 1 h, along with the significantly reduced self-digestion of trypsin and the improved reproducibility on the identification of proteins by the mass spectrometry-based proteomic approach. It showed that the number of identified bovine serum (BS) proteins on the commercial Fe3O4 nanoparticles was increased by 13% for the immobilized trypsin with 1 h digestion as compared to that of using free trypsin with even overnight digestion. In addition, the on-bead digestion of using the immobilized trypsin was further applied on the identification of human plasma protein corona on the commercial Fe3O4 nanoparticles, which leads the efficient digestion of the human plasma proteins and the identification of 149 human plasma proteins corresponding to putative critical pathways and biological processes.
Co-reporter:Yongsheng Ji, Zhichao Xiong, Guang Huang, Jing Liu, Zhang Zhang, Zheyi Liu, Junjie Ou, Mingliang Ye and Hanfa Zou
Analyst 2014 vol. 139(Issue 19) pp:4987-4993
Publication Date(Web):23 Jul 2014
DOI:10.1039/C4AN00971A
Selective enrichment of glycopeptides from complicated biological samples is critical for glycoproteomics to obtain the structure and glycosylation information of glycoproteins using mass spectrometry (MS), which still remains a great challenge. Hydrophilic interaction chromatography (HILIC)-based strategies have been proposed for selective isolation of glycopeptides via the interactions between the glycan of glycopeptides and the matrices. However, the application of these methods is limited by the medium selectivity of HILIC matrices. In this study, hydrophilic metal–organic frameworks (MOFs) were fabricated and used as a HILIC matrix. The cross-linked CD-MOFs (LCD-MOFs) were facilely prepared with γ-cyclodextrin as ligand and possessed nano-sized cubic structure, superior hydrophilicity, and bio-compatibility. The LCD-MOFs performance for the selective enrichment of glycopeptides from the complex biological samples were investigated with a digested mixture of human immunoglobulin G (IgG) that was used as standard samples. In the selectivity assessment, the non-glycopeptides causing ion suppression to the glycopeptides were effectively removed, the signal of glycopeptides were enhanced significantly by LCD-MOFs, and twenty glycopeptides were identified with 67 fmol of IgG digest. In addition, the resulting LCD-MOFs demonstrated the lower detection limit (3.3 fmol) with a satisfactory recovery yield (84–103%) for glycopeptide enrichment from a digest of IgG. Furthermore, a promising protocol was developed for the selective enrichment of glycopeptides from mouse liver, and 344 unique N-glycosylation sites that mapped to 290 different glycoproteins were identified in a single MS run. The results clearly demonstrated that when used in a HILIC matrix, LCD-MOFs have great potential for identifying and enriching low-abundant glycopeptides in complex biological samples.
Co-reporter:Junfeng Huang, Fangjun Wang, Mingliang Ye, Hanfa Zou
Journal of Chromatography A 2014 Volume 1372() pp:1-17
Publication Date(Web):12 December 2014
DOI:10.1016/j.chroma.2014.10.107
•Major protein post-translational modifications (PTMs) and their enrichment strategies.•The challenges encountered in the current PTMs enrichment methods.•Separation strategies of PTM peptides for large scale PTMs analysis.Comprehensive analysis of the post-translational modifications (PTMs) on proteins at proteome level is crucial to elucidate the regulatory mechanisms of various biological processes. In the past decades, thanks to the development of specific PTM enrichment techniques and efficient multidimensional liquid chromatography (LC) separation strategy, the identification of protein PTMs have made tremendous progress. A huge number of modification sites for some major protein PTMs have been identified by proteomics analysis. In this review, we first introduced the recent progresses of PTM enrichment methods for the analysis of several major PTMs including phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and oxidation/reduction status. We then briefly summarized the challenges for PTM enrichment. Finally, we introduced the fractionation and separation techniques for efficient separation of PTM peptides in large-scale PTM analysis.
Co-reporter:Yangyang Bian, Chunxia Song, Kai Cheng, Mingming Dong, Fangjun Wang, Junfeng Huang, Deguang Sun, Liming Wang, Mingliang Ye, Hanfa Zou
Journal of Proteomics 2014 Volume 96() pp:253-262
Publication Date(Web):16 January 2014
DOI:10.1016/j.jprot.2013.11.014
•An enzyme assisted RP-RPLC approach was developed with high orthogonality.•Identifications from TripleTOF 5600 and Orbitrap Velos were highly complementary.•22446 sites corresponding to 6526 phosphoproteins were identified from human liver.•The largest phosphoproteome dataset of human liver were constructed.•Many novel phosphorylation sites in the metabolic enzymes were identified.Protein phosphorylation is one of the most common post-translational modifications. It plays key roles in regulating diverse biological processes of liver tissues. To better understand the role of protein phosphorylation in liver functions, it is essential to perform in-depth phosphoproteome analysis of human liver. Here, an enzyme assisted reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach with both RPLC separations operated with optimized acidic mobile phase was developed. High orthogonal separation was achieved by trypsin digestion of the Glu-C generated peptides in the fractions collected from the first RPLC separation. The phosphoproteome coverage was further improved by using two types of instruments, i.e. TripleTOF 5600 and LTQ Orbitrap Velos. A total of 22,446 phosphorylation sites, corresponding to 6526 nonredundant phosphoproteins were finally identified from normal human liver tissues. Of these sites, 15,229 sites were confidently localized with Ascore ≥ 13. This dataset was the largest phosphoproteome dataset of human liver. It can be a public resource for the liver research community and holds promise for further biology studies.Biological significanceThe enzyme assisted approach enabled the two RPLC separations operated both with optimized acidic mobile phases. The identifications from TripleTOF 5600 and Orbitrap Velos are highly complementary. The largest phosphoproteome dataset of human liver was generated.
Co-reporter:Hao Wan, Jinan Li, Wenguang Yu, Zheyi Liu, Quanqing Zhang, Weibing Zhang and Hanfa Zou
RSC Advances 2014 vol. 4(Issue 86) pp:45804-45808
Publication Date(Web):12 Sep 2014
DOI:10.1039/C4RA08692A
A yolk–shell nanocomposite composed of a magnetic mesoporous anatase TiO2 (Fe3O4@mTiO2) core, a medium cavity and an outermost mesoporous silica (mSiO2) shell was successfully fabricated. Due to a combination of the strong magnetic response, improved diffusion of peptides, numerous affinity sites towards phosphopeptides and the size-exclusion effect, the nanocomposite demonstrated high enrichment efficacy and selectivity towards endogenous phosphopeptides from human serum.
Co-reporter:Yi Zhang;Zhengyan Hu;Guiju Xu;Chuanzhou Gao;Ren’an Wu
Nano Research 2014 Volume 7( Issue 8) pp:1103-1115
Publication Date(Web):2014 August
DOI:10.1007/s12274-014-0473-4
Co-reporter:Jing Liu;Fangjun Wang;Jun Zhu;Jiawei Mao
Analytical and Bioanalytical Chemistry 2014 Volume 406( Issue 13) pp:3103-3109
Publication Date(Web):2014 May
DOI:10.1007/s00216-014-7716-9
Conventional N-glycoproteome analysis usually applies C18 reversed-phase (RP) adsorbent for sample purification, which will lead to unavoidable sample loss due to the high hydrophilicity of N-glycopeptides. In this study, a porous graphitized carbon (PGC) absorbent was combined with a C18 adsorbent for N-glycopeptide purification in comprehensive N-glycoproteome analysis based on the hydrophobic and polar interactions between carbon and N-glycans. It was observed that the small hydrophilic N-glycopeptides that cannot retain onto C18 adsorbent can be captured by the graphitized carbon, while the large hydrophobic N-glycopeptides that cannot retain onto the graphitized carbon can be feasibly captured by the C18 adsorbent. Comparing with sample purification by using C18 adsorbent only, 28.5 % more N-glycopeptides were identified by combining both C18 and PGC adsorbents. The C18-PGC strategy was further applied for both sample purification and pre-fractionation of a complex protein sample from HeLa cell. After hydrophilic interaction chromatography enrichment, 1,484 unique N-glycopeptides with 1,759 unique N-glycosylation sites were finally identified.
Co-reporter:Yanbo Pan;Kai Cheng;Jiawei Mao;Fangjie Liu
Analytical and Bioanalytical Chemistry 2014 Volume 406( Issue 25) pp:6247-6256
Publication Date(Web):2014 October
DOI:10.1007/s00216-014-8071-6
Trypsin is the popular protease to digest proteins into peptides in shotgun proteomics, but few studies have attempted to systematically investigate the kinetics of trypsin-catalyzed protein digestion in proteome samples. In this study, we applied quantitative proteomics via triplex stable isotope dimethyl labeling to investigate the kinetics of trypsin-catalyzed cleavage. It was found that trypsin cleaves the C-terminal to lysine (K) and arginine (R) residues with higher rates for R. And the cleavage sites surrounded by neutral residues could be quickly cut, while those with neighboring charged residues (D/E/K/R) or proline residue (P) could be slowly cut. In a proteome sample, a huge number of proteins with different physical chemical properties coexists. If any type of protein could be preferably digested, then limited digestion could be applied to reduce the sample complexity. However, we found that protein abundance and other physicochemical properties, such as molecular weight (Mw), grand average of hydropathicity (GRAVY), aliphatic index, and isoelectric point (pI) have no notable correlation with digestion priority of proteins.
Co-reporter:Zhengyan Hu, Hongyan Zhang, Yi Zhang, Ren’an Wu, Hanfa Zou
Colloids and Surfaces B: Biointerfaces 2014 Volume 121() pp:354-361
Publication Date(Web):1 September 2014
DOI:10.1016/j.colsurfb.2014.06.016
•The determination of human plasma protein coronas on Fe3O4 nanoparticles with different particle sizes was carried out.•Particles size impacts the protein compositions of plasma coronas formed on Fe3O4 nanoparticles.•Particles size affects the protein abundances of plasma protein coronas on Fe3O4 nanoparticles.When nanoparticles (NPs) enter into biological systems, proteins would interact with NPs to form the protein corona that can critically impact the biological identity of the nanomaterial. Owing to their fundamental scientific interest and potential applications, Fe3O4 NPs of different sizes have been developed for applications in cell separation and protein separation and as contrast agents in magnetic resonance imaging (MRI), etc. Here, we investigated whether nanoparticle size affects the formation of protein coronas around Fe3O4 NPs. Both the identification and quantification results demonstrated that particle size does play an important role in the formation of plasma protein coronas on Fe3O4 NPs; it not only influenced the protein composition of the formed plasma protein corona but also affected the abundances of the plasma proteins within the coronas. Understanding the different binding profiles of human plasma proteins on Fe3O4 NPs of different sizes would facilitate the exploration of the bio-distributions and biological fates of Fe3O4 NPs in biological systems.
Co-reporter:Dr. Zhichao Xiong ;Dr. Yongsheng Ji ;Chunli Fang;Quanqing Zhang; Lingyi Zhang; Mingliang Ye; Weibing Zhang ; Hanfa Zou
Chemistry - A European Journal 2014 Volume 20( Issue 24) pp:7389-7395
Publication Date(Web):
DOI:10.1002/chem.201400389
Abstract
Facile preparation of core–shell magnetic metal–organic framework nanospheres by a layer-by-layer approach is presented. The nanospheres have high surface area (285.89 cm2 g−1), large pore volume (0.18 cm3 g−1), two kinds of mesopores (2.50 and 4.72 nm), excellent magnetic responsivity (55.65 emu g−1), structural stability, and good dispersibility. The combination of porosity, hydrophobicity, and uniform magnetism was exploited for effective enrichment of peptides with simultaneous exclusion of high molecular weight proteins. The nanospheres were successfully applied in the selective enrichment of endogenous peptides in human serum.
Co-reporter:Hao Wan, Hongqiang Qin, Zhichao Xiong, Weibing Zhang and Hanfa Zou
Nanoscale 2013 vol. 5(Issue 22) pp:10936-10944
Publication Date(Web):29 Aug 2013
DOI:10.1039/C3NR02932H
Magnetic mesoporous carbon microspheres with a yolk–shell structure (YSMMCS) have been prepared via a new in situ carbon source strategy. The material was fabricated by two shells coated onto the Fe3O4 particles; the inner dense and thick silica shell could protect the magnetic core from harsh acidic solvents as well as induce the void between the core and the outer shell for the yolk–shell structure, while the outer organosilica shell was used as the template and carbon source for in situ preparation of a carbon shell with mesoporous structure. A C18-alkyl chain was incorporated in situ as the carbon precursor efficiently, avoiding the conventional infiltration step, which was very difficult to manipulate and time-consuming with the possibility of losing the carbon precursor. The resulting yolk–shell magnetic mesoporous carbon microspheres exhibited a high surface area (273.15 m2 g−1), a large pore volume (0.31 cm3 g−1), and a strong magnetic response (a saturation magnetization value of 34.57 emu g−1). As a result of the void between the core and the outer shell and the π–π stacking effect, adsorption capacity reached 191.64 mg g−1 by using Rhodamine B as a standard analyte, indicating the great potential application of the material as drug carriers. Owing to the inherent hydrophobicity and high surface area, the composite material showed better performance in the enrichment of peptides than a magnetic mesoporous silica material (Fe2O3@nSiO2@mSiO2). According to the LC-MS/MS results, about 51 and 29 nonredundant peptides were identified from tryptic digests of 5 nM BSA. Additionally, taking advantage of the mesoporous structure and strong magnetic response, the material was utilized to selectively extract low abundance endogenous peptides from human serum in the presence of high abundance proteins. Based on the LC-MS/MS results, 962 endogenous peptides were obtained by 2.5 mg YSMMCS relative to 539 endogenous peptides by 5 mg Fe2O3@nSiO2@mSiO2, confirming the outstanding performance of YSMMCS in peptidome analysis.
Co-reporter:Zhichao Xiong, Hongqiang Qin, Hao Wan, Guang Huang, Zhang Zhang, Jing Dong, Lingyi Zhang, Weibing Zhang and Hanfa Zou
Chemical Communications 2013 vol. 49(Issue 81) pp:9284-9286
Publication Date(Web):29 Aug 2013
DOI:10.1039/C3CC45008B
Magnetic nanoparticles (MNPs) coated with multilayer polysaccharide shells have been fabricated using a layer-by-layer approach via the alternate deposition of hyaluronan (HA) and chitosan (CS) onto the surface, and the hydrophilic materials were utilized for effective and selective enrichment of glycopeptides in biological samples.
Co-reporter:Hongqiang Qin, Zhengyan Hu, Fangjun Wang, Yi Zhang, Liang Zhao, Guiju Xu, Ren'an Wu and Hanfa Zou
Chemical Communications 2013 vol. 49(Issue 45) pp:5162-5164
Publication Date(Web):15 Apr 2013
DOI:10.1039/C3CC41479E
Silica–carbon composite nanoparticles (NP-MCM-C) with uniform shapes and highly ordered mesoporous structures are directly prepared by using template polymers as the carbon source. And, taking advantage of the size exclusion effect of the mesopores to proteins and the specific interaction between carbon and oligosaccharides, the prepared nanoparticles are utilized to enrich N-linked glycans from complex biological samples with high selectivity and efficiency.
Co-reporter:Jing Liu, Fangjun Wang, Hui Lin, Jun Zhu, Yangyang Bian, Kai Cheng, and Hanfa Zou
Analytical Chemistry 2013 Volume 85(Issue 5) pp:2847
Publication Date(Web):February 5, 2013
DOI:10.1021/ac400315n
Despite the importance of protein N-glycosylation in a series of biological processes, in-depth characterization of protein glycosylation is still a challenge due to the high complexity of biological samples and the lacking of highly sensitive detection technologies. We developed a monolithic capillary column based glycoproteomic reactor enabling high-sensitive mapping of N-glycosylation sites from minute amounts of sample. Unlike the conventional proteomic reactors with only strong-cation exchange or hydrophilic-interaction chromatography columns, this novel glycoproteomic reactor was composed of an 8 cm long C12 hydrophobic monolithic capillary column for protein digestion and a 6 cm long organic–silica hybrid hydrophilic monolithic capillary column for glycopeptides enrichment and deglycosylation, which could complete whole-sample preparation including protein purification/desalting, tryptic digestion, enrichment, and deglycosylation of glycopeptides within about 3 h. The developed reactor exhibited high detection sensitivity in mapping of N-glycosylation sites by detection limit of horseradish peroxidase as low as 2.5 fmol. This reactor also demonstrated the ability in complex sample analysis, and in total, 486 unique N-glycosylation sites were reliably mapped in three replicate analyses of a protein sample extracted from ∼104 HeLa cells.
Co-reporter:Yi Zhang, Zhengyan Hu, Hongqiang Qin, Fangjie Liu, Kai Cheng, Ren’an Wu, and Hanfa Zou
Analytical Chemistry 2013 Volume 85(Issue 15) pp:7038
Publication Date(Web):July 1, 2013
DOI:10.1021/ac401269g
Since nanoparticles could be ingested by cells naturally and target at a specific cellular location as designed, the extraction of intracellular proteins from living cells for large-scale analysis by nanoprobes seems to be ideally possible. Nucleic acid associated proteins (NAaP) take the crucial position during biological processes in maintaining and regulating gene structure and gene related behaviors, yet there are still challenges during the global investigation of intracellular NAaP, especially from living cells. In this work, a strategy to extract intracellular proteins from living cells with the magnetic carbon nanotube (oMWCNT@Fe3O4) as an intracellular probe is developed, to achieve the high throughput analysis of NAaP from living human hepatoma BEL-7402 cells with a mass spectrometry-based proteomic approach. Due to the specific intracellular localization of the magnetic carbon nanotubes around nuclei and its strong interaction with nucleic acids, the highly efficient extraction was realized for cellular NAaP from living cells, with the capability of identifying 2383 intracellular NAaP from only ca. 10 000 living cells. This method exhibited potential applications in dynamic and in situ analysis of intracellular proteins.
Co-reporter:Fangjie Liu, Mingliang Ye, Chunli Wang, Zhengyan Hu, Yi Zhang, Hongqiang Qin, Kai Cheng, and Hanfa Zou
Analytical Chemistry 2013 Volume 85(Issue 15) pp:7024
Publication Date(Web):July 16, 2013
DOI:10.1021/ac4017693
Trypsin was immobilized on a variety of materials to improve digestion efficiency. However, because the immobilized trypsin will digest proteins during electrophoresis, direct immobilization of active trypsin in polyacrylamide gel will compromise the protein separation. To overcome this problem, here we report a novel polyacrylamide gel with switchable trypsin activity. It was prepared by copolymerization of the PEG–trypsin–aprotinin complex during the gel-casting step. Because the inhibitor aprotinin binds strongly with trypsin at alkaline pH, this novel gel does not display hydrolytic activity during electrophoresis. After electrophoresis, the activity of trypsin embedded in gel could be recovered by simply washing away the bound inhibitor at a low pH. It was demonstrated that this unique switchable activity design allowed high resolution of the complex protein mixture during electrophoresis and highly efficient digestion of the separated proteins in situ in the gel after electrophoresis.
Co-reporter:Junjie Ou, Zhenbin Zhang, Hui Lin, Jing Dong, Hanfa Zou
Analytica Chimica Acta 2013 Volume 761() pp:209-216
Publication Date(Web):25 January 2013
DOI:10.1016/j.aca.2012.11.052
A simple approach to fabricate hybrid monolithic column within the confines of fused-silica capillaries (75 μm i.d.) was introduced. A polyhedral oligomeric silsesquioxanes (POSS) reagent containing a methacrylate group was selected as functional monomer, and copolymerized with bisphenol A dimethacrylate (BPADMA) or ethylene dimethacrylate (EDMA) in the presence of porogenic solvents via thermally initiated free radical polymerization. After optimization of the preparation conditions, two POSS-containing hybrid monoliths were successfully prepared and exhibited good permeability and stability. By comparison of the separation efficiencies of the resulting poly(POSS-co-BPADMA) and poly(POSS-co-EDMA) monoliths in capillary electrochromatography (CEC) and capillary liquid chromatography (cLC), it was indicated the former has better column efficiencies for alkylbenzenes, phenols, anilines and PAHs in CEC and cLC than the latter. Particularly, the hybrid poly(POSS-co-BPADMA) monolith is more suitable for separation of PAHs due to π–π interaction between the analytes and aromatic rings in the surface of monolithic stationary phase.Graphical abstract.Highlights► A POSS reagent was selected as monomer to prepare hybrid monolithic column. ► Two resulting hybrid monoliths were successfully applied for CEC and cLC. ► The hybrid poly(POSS-co-BPADMA) monolith exhibits better selectivity for PAHs.
Co-reporter:Hui Lin, Junjie Ou, Shouwan Tang, Zhenbin Zhang, Jing Dong, Zhongshan Liu, Hanfa Zou
Journal of Chromatography A 2013 Volume 1301() pp:131-138
Publication Date(Web):2 August 2013
DOI:10.1016/j.chroma.2013.05.069
•A POSS-based functionalizable hybrid monolith was successfully prepared via ring-opening polymerization.•The obtained hybrid monolith possesses well-controlled 3D skeleton.•A column efficiency of 110,000 N/m was achieved for isocratic separation of alkylbenzenes.•The obtained hybrid monolith showed high stability and good surface tailorability.An organic–inorganic hybrid monolith was prepared by a single-step ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS) with poly(ethylenimine) (PEI). The obtained hybrid monoliths possessed high ordered 3D skeletal microstructure with dual retention mechanism that exhibits reversed-phase (RP) mechanism under polar mobile phase and hydrophilic-interaction liquid chromatography (HILIC) retention mechanism under less polar mobile phase. The high column efficiencies of 110,000 N/m can be achieved for separation of alkylbenzenes in capillary reversed-phase liquid chromatography (cLC). Due to the robust property of hybrid monolith and the rich primary and secondary amino groups on its surface, the resulting hybrid monolith was easily modified with γ-gluconolactone and physically coated with cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC), respectively. The former was successfully applied for HILIC separation of neutral, basic and acidic polar compounds as well as small peptides, and the latter for enantioseparation of racemates in cLC. The high column efficiencies were achieved in all of those separations. These results demonstrated that the hybrid monolith (POSS–PEI) possessed high stability and good surface tailorbility, potentially being applied for other research fields
Co-reporter:Xu Wang, Yangyang Bian, Kai Cheng, Li-Fei Gu, Mingliang Ye, Hanfa Zou, Samuel Sai-Ming Sun, Jun-Xian He
Journal of Proteomics 2013 Volume 78() pp:486-498
Publication Date(Web):14 January 2013
DOI:10.1016/j.jprot.2012.10.018
Large-scale protein phosphorylation analysis by MS is emerging as a powerful tool in plant signal transduction research. However, our current understanding of the phosphorylation regulatory network in plants is still very limited. Here, we report on a proteome-wide profiling of phosphopeptides in nine-day-old Arabidopsis (Arabidopsis thaliana) seedlings by using an enrichment method combining the titanium (Ti4 +)-based IMAC and the RP-strong cation exchange (RP-SCX) biphasic trap column-based online RPLC. Through the duplicated RPLC–MS/MS analyses, we identified 5348 unique phosphopeptides for 2552 unique proteins. Among the phosphoproteins identified, 41% of them were first-time identified. Further evolutionary conservation and phosphorylation motif analyses of the phosphorylation sites discovered 100 highly conserved phosphorylation residues and identified 17 known and 14 novel motifs specific for Ser/Thr protein kinases. Gene ontology and pathway analyses revealed that many of the new identified phosphoproteins are important regulatory proteins that are involved in diverse biological processes, particularly in central metabolisms and cell signaling. Taken together, our results provided not only new insights into the complex phosphoregulatory network in plants but also important resources for future functional studies of protein phosphorylation in plant growth and development.Highlights► Identified 2552 unique phosphoproteins and 41% of them are first reported ► Identified 17 known and 14 novel phosphorylation motifs specific for Ser/Thr kinases ► Discovered 100 highly conserved phosphorylation residues ► Provide new insights into plant phosphorylation regulatory networks
Co-reporter:Zheyi Liu, Junjie Ou, Zhongshan Liu, Jing Liu, Hui Lin, Fangjun Wang, Hanfa Zou
Journal of Chromatography A 2013 Volume 1317() pp:138-147
Publication Date(Web):22 November 2013
DOI:10.1016/j.chroma.2013.09.004
•The separation of intact proteins on the POSS-based hybrid monolithic capillary column was investigated.•The POSS-based monolithic capillary columns exhibits good permeability and can be operated at high flow rate with slight decrease of the column efficiency.•Fast separation of seven intact proteins can be realized in 2.5 min.High-efficient separation of intact proteins is still a huge challenge in proteome analysis of complex biological samples by using capillary columns. In this study, four POSS-based hybrid monolithic capillary columns were prepared and applied in nano-flow liquid chromatography (Nano-LC) separation of intact proteins. It was observed that the POSS-based hybrid monolithic columns exhibit high permeability, good LC separation reproducibility and column efficiency for intact protein separation. The effects of different LC separation conditions such as flow rate, gradient steepness, column length and mobile phase additives on the LC separation efficiency of the POSS-based hybrid monolithic column were systematically examined. Finally, fast LC separation of 7 proteins mixture was realized in 2.5 min by using the optimized conditions on the 100 μm i.d. POSS-based hybrid monolithic capillary column.
Co-reporter:Jun Zhu, Fangjun Wang, Kai Cheng, Chunxia Song, Hongqiang Qin, Lianghai Hu, Daniel Figeys, Mingliang Ye, Hanfa Zou
Journal of Proteomics 2013 Volume 78() pp:389-397
Publication Date(Web):14 January 2013
DOI:10.1016/j.jprot.2012.10.006
As human serum is an important source for early diagnosis of many serious diseases, analysis of serum proteome and peptidome has been extensively performed. However, the serum phosphopeptidome was less explored probably because the effective method for database searching is lacking. Conventional database searching strategy always uses the whole proteome database, which is very time-consuming for phosphopeptidome search due to the huge searching space resulted from the high redundancy of the database and the setting of dynamic modifications during searching. In this work, a focused database searching strategy using an in-house collected human serum pro-peptidome target/decoy database (HuSPep) was established. It was found that the searching time was significantly decreased without compromising the identification sensitivity. By combining size-selective Ti (IV)-MCM-41 enrichment, RP–RP off-line separation, and complementary CID and ETD fragmentation with the new searching strategy, 143 unique endogenous phosphopeptides and 133 phosphorylation sites (109 novel sites) were identified from human serum with high reliability.Highlights► Searching space was greatly reduced by a focused database searching strategy. ► Human serum phosphopeptidome was extensively analyzed. ► Largest number of serum endogenous phosphopeptides were confidently identified.
Co-reporter:Kai Li, Zhen Sun, Jianyong Zheng, Yuanyuan Lu, Yangyang Bian, Mingliang Ye, Xin Wang, Yongzhan Nie, Hanfa Zou, Daiming Fan
Journal of Proteomics 2013 Volume 82() pp:130-140
Publication Date(Web):26 April 2013
DOI:10.1016/j.jprot.2013.02.021
•We study the MDR related cell surface N-glycoproteome in gastric cancer.•We obtain 11 N-glycoproteins as potential biomarkers for MDR in gastric cancer.•N-glycosylation at Asn99 is essential for P-gp function in MDR of gastric cancer.•We report a modified method for screening malignancy related N-glycoproteins.Human gastric cancer is a big public health problem. Multidrug resistance is a main obstacle to successful chemotherapeutic treatment in gastric cancers and the underlying mechanism is not clear. Glycosylation, one of the most important post translational modifications of proteins, plays a vital role in diverse aspects of tumor progression. In the present study, we applied two multidrug resistance cell lines and their parental drug sensitive gastric cancer cell line to a modified cell surface capturing strategy with triplex labeling to characterize MDR related cell surface glycoproteome. Finally, 56 cell membrane glycoproteins were successfully identified via combination of identification by glycopeptides and quantitation by non-glycopeptides, and 11 of them were found to be differentially expressed with the same trend in both drug resistant cell lines compared with that in sensitive cell line. The further analysis by western blot and in vitro drug sensitivity assay demonstrated that our approach is reliable and accurate and suggested that these glycoproteins may represent as biomarkers for multidrug resistance in gastric cancer.Biological significanceIn this study, we performed a cell surface glycoproteomics research of multidrug resistance in gastric cancer using a modified CSC approach. Totally we identified and quantified 11 membrane N-glycoproteins which were significantly changed in MDR gastric cancer cells. These glycoproteins are quite possible to be biomarkers for predicting MDR or key regulators for targeted therapy, and are also helpful for better interpreting the sophisticated mechanisms of MDR in gastric cancer. In addition to that, this approach used in this study can be well applied to screen aberrantly glycosylated biomarkers associated with other malignant phenotypes of various kinds of cancers.
Co-reporter:Hui Lin;Zhenbin Zhang;Jing Dong;Zhongshan Liu;Junjie Ou
Journal of Separation Science 2013 Volume 36( Issue 17) pp:2819-2825
Publication Date(Web):
DOI:10.1002/jssc.201300417
A new organic–inorganic hybrid monolith was prepared by the ring-opening polymerization of octaglycidyldimethylsilyl polyhedral oligomeric silsesquioxane (POSS) with 1,4-butanediamine (BDA) using 1-propanol, 1,4-butanediol, and PEG 10 000 as a porogenic system. Benefiting from the moderate phase separation process, the resulting poly(POSS-co-BDA) hybrid monolith possessed a uniform microstructure and exhibited excellent performance in chromatographic applications. Neutral, acidic, and basic compounds were successfully separated on the hybrid monolith in capillary LC (cLC), and high column efficiencies were achieved in all of the separations. In addition, as the amino groups could generate a strong EOF, the hybrid monolith was also applied in CEC for the separation of neutral and polar compounds, and a satisfactory performance was obtained. These results demonstrate that the poly(POSS-co-BDA) hybrid monolith is a good separation media in chromatographic separations of various types of compounds by both cLC and CEC.
Co-reporter:Bo Xu, Chen Chen, Fangjun Wang, Yangyang Bian, Kai Cheng, Hongqiang Qin, Chunxia Song, Jun Zhu, Jing Liu, Mingliang Ye and Hanfa Zou
Analytical Methods 2013 vol. 5(Issue 12) pp:2939-2946
Publication Date(Web):22 Mar 2013
DOI:10.1039/C3AY00064H
The titanium silicalite-1 (TS-1) with post-treatment of chemical selective desilication (T-TS-1) has been synthesized, characterized and applied as a potential adsorbent for selective capture of phosphopeptides from complex biological samples prior to mass spectrometry analysis. A T-TS-1 material-based successive phosphopeptide enrichment strategy has also been established for real biological sample analysis, which exhibited high enrichment selectivity and is comparable to existing materials.
Co-reporter:Jing LIU, Fang-Jun WANG, Zhen-Bin ZHANG, Han-Fa ZOU
Chinese Journal of Analytical Chemistry 2013 Volume 41(Issue 1) pp:10-14
Publication Date(Web):January 2013
DOI:10.1016/S1872-2040(13)60619-2
We developed a reversed-phase monolithic column based enzyme reactor for protein analysis. The effects of buffer solution, porous structure of the column, digestion time and hydrophobicity of solid phase on the digestion performance of enzyme reactor were systematically investigated in terms of the number of identified unique peptides, sequence coverage and the level of missed cleavage as the evaluation indicators. The results demonstrated that the reversed-phase monolithic column based enzyme reactor had high digestion efficiency and might be further extended to the proteome analysis of complex samples.Base peak chromatogram for separation of 1 μg BSA digest by C12 monolithic column microreactor. This new enzyme reactor showed more base peaks and higher peak intensity in the peak chromatogram compared with C18 monolithic column and C18 packed column microreactors.
Co-reporter:Zhenbin Zhang;Fangjun Wang;Junjie Ou;Hui Lin
Analytical and Bioanalytical Chemistry 2013 Volume 405( Issue 7) pp:2265-2271
Publication Date(Web):2013 March
DOI:10.1007/s00216-012-6589-z
A butyl–silica hybrid monolithic column for bioseparation by capillary liquid chromatography (cLC) was prepared with butyl methacrylate and alkoxysilanes through a “one-pot” process. The effects of polycondensation temperature, volume percentage of N,N′-dimethylformamide, and content of cetyltrimethylammonium bromide and butyl methacrylate on the morphologies of the hybrid monolithic columns prepared were investigated in detail. Baseline separations of proteins and small peptides on the hybrid monolithic column were achieved by cLC with gradient elution. In addition, the resulting hybrid column was also applied for analysis of tryptic digests of bovine serum albumin by cLC coupled with tandem mass spectrometry. The results demonstrate its potential application in separation of complex biological samples.
Co-reporter:Yanbo Pan;Dr. Mingliang Ye;Dr. Liang Zhao;Kai Cheng;Mingming Dong;Chunxia Song;Hongqiang Qin;Dr. Fangjun Wang;Dr. Hanfa Zou
Angewandte Chemie International Edition 2013 Volume 52( Issue 35) pp:9205-9209
Publication Date(Web):
DOI:10.1002/anie.201303429
Co-reporter:Liang Zhao, Hongqiang Qin, Zhengyan Hu, Yi Zhang, Ren'an Wu and Hanfa Zou
Chemical Science 2012 vol. 3(Issue 9) pp:2828-2838
Publication Date(Web):19 Jun 2012
DOI:10.1039/C2SC20363D
Immobilized metal affinity chromatography (IMAC) is a powerful method in phosphopeptide enrichment. However, the achievement of highly specific enrichment and sensitive detection of phosphopeptide by IMAC is still a big challenge because of the lack of high specificity and large binding capacity of conventional IMAC materials. Here, we report a novel IMAC nanoparticle to dramatically improve the enrichment specificity for the phosphopeptide by introducing a titanium phosphate moiety on a poly(ethylene glycol) methacrylate (PEG) brush decorated Fe3O4@SiO2 core–shell nanoparticle (denoted as Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle). The thicker grafting layer of the PEG brushes has a higher chelating capacity of titanium ions. Due to the combination of the superior nonfouling property and the enhanced binding capacity of the grafted PEG brushes, the Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle demonstrated a high phosphopeptide recovery (over 70%) and low limit of detection (0.5 fmol), along with an exceptional great specificity to capture phosphopeptides from a tryptic digest of the mixture of a nonphosphorylated protein BSA and a phosphorylated protein α-casein with molar ratios of BSA/α-casein up to 2000:1. In the analysis of a real complex biological sample, the tryptic digests of Arabidopsis, 2447 unique phosphopeptides have been identified, showing a superior performance of the Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle than that of Fe3O4@SiO2–Ti4+ (1186) and commercial TiO2 microspheres (961). We believe that the PEG decoration for IMAC materials will be a convenient approach to significantly improve the specificity and the binding capacity of phosphopeptide enrichment.
Co-reporter:Hongqiang Qin, Fangjun Wang, Peiyuan Wang, Liang Zhao, Jun Zhu, Qihua Yang, Ren'an Wu, Mingliang Ye and Hanfa Zou
Chemical Communications 2012 vol. 48(Issue 7) pp:961-963
Publication Date(Web):03 Nov 2011
DOI:10.1039/C1CC15222J
Ti4+-EPO nanoparticles were adopted as the adsorbent for in situ solid phase enrichment and isotope labeling of endogenous phosphopeptides, which has great potential application in high-throughput analyses of biological samples for screening and discovery of disease-specific biomarkers.
Co-reporter:Zhichao Xiong, Liang Zhao, Fangjun Wang, Jun Zhu, Hongqiang Qin, Ren'an Wu, Weibing Zhang and Hanfa Zou
Chemical Communications 2012 vol. 48(Issue 65) pp:8138-8140
Publication Date(Web):25 Jun 2012
DOI:10.1039/C2CC33600F
Hybrid Fe3O4@SiO2@PEG-Maltose MNPs were synthesized by SI-ATRP of branched PEG brushes on the surface and subsequent functionalization with hydrophilic maltose group, and the multifunctional materials were utilized for selective enrichment of N-linked glycopeptides from biological samples with high specifity, high sensitivity, and large binding capacity.
Co-reporter:Hongqiang Qin, Fangjun Wang, Yi Zhang, Zhengyan Hu, Chunxia Song, Ren'an Wu, Mingliang Ye and Hanfa Zou
Chemical Communications 2012 vol. 48(Issue 50) pp:6265-6267
Publication Date(Web):01 May 2012
DOI:10.1039/C2CC31705B
Highly site-selective dimethyl labeling of N-terminus of peptides has been obtained by adjusting the acidic strength of the reaction solution. And this selective labeling strategy combined with the use of different isotope formaldehyde reagents has been successfully used in the proteome quantification by using the isobaric peptide cross-sequence labeling method, which would play increasingly important roles in the clinical diagnosis, especially in the discovery of biomarkers for diseases.
Co-reporter:Bo Xu, Lipeng Zhou, Fangjun Wang, Hongqiang Qin, Jun Zhu and Hanfa Zou
Chemical Communications 2012 vol. 48(Issue 12) pp:1802-1804
Publication Date(Web):06 Dec 2011
DOI:10.1039/C2CC16662C
Hierarchical Ti-aluminophosphate-5 molecular sieves templated by glucose have been synthesized and applied as a potential adsorbent for the first time to selectively capture phosphopeptides from complex peptide mixtures prior to MALDI-TOF MS analysis.
Co-reporter:Hui Lin, Junjie Ou, Zhenbin Zhang, Jing Dong, Minghuo Wu, and Hanfa Zou
Analytical Chemistry 2012 Volume 84(Issue 6) pp:2721-2728
Publication Date(Web):February 22, 2012
DOI:10.1021/ac3001429
A simple single-step thermal-treatment “one-pot” approach for the preparation of organic-silica hybrid capillary monolithic columns is described. In this improved method, the cross-linker vinyltrimethoxysilane (VTMS) was replaced by 3-methacryloxypropyltrimethoxysilane (γ-MAPS), which is more active in polymerization reactions, and only one thermal treatment step was required in the preparation of hybrid monoliths. Two zwitterionic organic-silica monolithic columns were successfully synthesized by using [2-(methacryloyloxy)ethyl]dimethyl-(3-sulfopropyl)ammonium hydroxide (MSA) and 2-methacryloyloxyethyl phosphorylcholine (MPC) as the organic monomers. The effects of the tetramethoxysilane (TMOS)/γ-MAPS molar ratio, content of monomer, composition of porogenic solvent, and reaction temperature on the morphologies of the hybrid monoliths were investigated. The MSA-silica and MPC-silica hybrid monolithic columns exhibited good permeability and good mechanical stability. The monolithic columns were used for the separation of polar compounds by capillary hydrophilic-interaction chromatography (cHILIC). A typical HILIC retention mechanism was observed at higher organic solvent contents (>50% ACN). The MSA monoliths were further investigated in the separation of various neutral, basic, and acidic analytes, as well as small peptides, by capillary liquid chromatography (cLC), and high efficiency and satisfactory reproducibility were achieved. In addition, the analysis of a tryptic digest of bovine serum albumin (BSA) by cLC tandem mass spectrometry (cLC-MS/MS) with an MSA monolith further demonstrated its potential in the separation of biological samples.
Co-reporter:Jun Zhu, Fangjun Wang, Rui Chen, Kai Cheng, Bo Xu, Zhimou Guo, Xinmiao Liang, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2012 Volume 84(Issue 11) pp:5146
Publication Date(Web):May 9, 2012
DOI:10.1021/ac3000732
Sample handling procedures including protein digestion, glycopeptide enrichment, and deglycosylation have significant impact on the performance of glycoproteome analysis. Several glycoproteomic analysis systems were developed to integrate some of these sample preparation procedures. However, no microsystem integrates all of above three procedures together. In this work, we developed a glycoproteomic microreactor enabling seamless integration of all these procedures. In this reactor, trypsin digestion was accelerated by adding acetonitrile to 80%, and after acidification of protein digest by trifluoroacetic acid (TFA), the following hydrophilic interaction chromatography (HILIC) enrichment and deglycosylation were sequentially performed without any desalting, lyophilization, or buffer exchange steps. The total processing time could be as short as 1.5 h. The detection limit of human IgG as low as 30 fmol was also achieved. When applied to human serum glycoproteome analysis, a total number of 92, 178, and 221 unique N-glycosylation sites were identified from three replicate analyses of 10 nL, 100 nL, and 1 μL of human serum, respectively. It was demonstrated that the glycoproteomic microreactor based method had very high sensitivity and was well suited for glycoproteome analysis of minute protein samples.
Co-reporter:Zhen Sun, Hongqiang Qin, Fangjun Wang, Kai Cheng, Mingming Dong, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2012 Volume 84(Issue 20) pp:8452
Publication Date(Web):September 25, 2012
DOI:10.1021/ac302130r
Incorporation of isotopic tag onto peptides via chemical labeling is a popular approach for quantitative proteomics. Chemical labeling via solution based methods usually lead to a tedious process and sample loss because several sample preparation steps including buffer exchange and desalting are performed. In this study, a solid phase based labeling approach by integration of glycopeptide enrichment and stable isotope labeling on hydrazide beads was developed for relative quantification of protein glycosylation, by which enrichment, washing, labeling, and release of the glycopeptides were all performed on the hydrazide beads sequentially. This approach was proved to be accurate in quantitative glycoproteome analysis and have good linearity range with 2 orders of magnitude for quantification of glycopeptides. Compared with dimethyl labeling conventionally performed in solution, the developed approach has better enrichment recovery (10–330% improvement) and high detection sensitivity in which 42% of annotated glycosites (vs 26%) still can be quantified using only 10 μg of four standard glycoprotein mixtures and 400 μg of bovine serum album interference as starting sample. The applicability of the approach for quantitative glycopeptide profiling was also explored by differential analysis of glycoproteome between human normal serum and liver cancer serum.
Co-reporter:Yi Zhang, Zhengyan Hu, Hongqiang Qin, Xiaoluan Wei, Kai Cheng, Fangjie Liu, Ren’an Wu, and Hanfa Zou
Analytical Chemistry 2012 Volume 84(Issue 23) pp:10454
Publication Date(Web):November 4, 2012
DOI:10.1021/ac302695u
Nucleic acid associated proteins (NAaP) play the essential roles in gene regulation and protein expression. The global analysis of cellular NAaP would give a broad insight to understand the interaction between nucleic acids and the associated proteins, such as the important proteinous regulation factors on nucleic acids. Proteomic analysis presents a novel strategy to investigate a group of proteins. However, the large scale analysis of NAaP is yet impossible due to the lack of approaches to harvest target protein groups with a high efficiency. Herein, a simple and efficient method was developed to collect cellular NAaP using magnetic oxidized carbon nanotubes based on the strong interaction between carbon nanotubes and nucleic acids along with corresponding associated proteins. We found that the magnetic oxidized carbon nanotubes demonstrated a nearly 100% extraction efficiency for intracellular nucleic acids from cells in vitro. Importantly, the proteins associated on nucleic acids could be highly efficiently harvested using magnetic oxidized carbon nanotubes due to the binding of NAaP on nucleic acids. 1594 groups of nuclear NAaP and 2595 groups of cellular NAaP were extracted and identified from about 1 000 000 cells, and 803 groups of NAaP were analyzed with only about 10 000 cells, showing a promising performance for the proteomic analysis of NAaP from minute cellular samples. This highly efficient extraction strategy for NAaP is a simple approach to identify cellular nucleic acid associated proteome, and we believed this strategy could be further applied in systems biology to understand the gene expression and regulation.
Co-reporter:Rui Chen, Fangjun Wang, Yexiong Tan, Zhen Sun, Chunxia Song, Mingliang Ye, Hongyang Wang, Hanfa Zou
Journal of Proteomics 2012 Volume 75(Issue 5) pp:1666-1674
Publication Date(Web):16 February 2012
DOI:10.1016/j.jprot.2011.12.015
Direct mass spectrometric analysis of aberrant protein glycosylation is a challenge to the current analytical techniques. Except lectin affinity chromatography, no other glycosylation enrichment techniques are available for analysis of aberrant glycosylation. In this study, we developed a combined chemical and enzymatic strategy as an alternative for the mass spectrometric analysis of aberrant glycosylation. Sialylated glycopeptides were enriched with reverse glycoblotting, cleaved by endoglycosidase F3 and analyzed by mass spectrometry with both neutral loss triggered MS3 in collision induced dissociation (CID) and electron transfer dissociation (ETD). Interestingly, a great part of resulted glycopeptides were found with fucose attached to the N-acetylglucosamine (N-GlcNAc), which indicated that the aberrant glycosylation that is carrying both terminal sialylation and core fucosylation was identified. Totally, 69 aberrant N-glycosylation sites were identified in sera samples from hepatocellular carcinoma (HCC) patients. Following the identification, quantification of the level of this aberrant glycosylation was also carried out using stable isotope dimethyl labeling and pooled sera sample from liver cirrhosis and HCC was compared. Six glycosylation sites demonstrated elevated level of aberrancy, which demonstrated that our developed strategy was effective in both qualitative and quantitative studies of aberrant glycosylation.Highlights► Isolation of N-glycosites with both terminal sialylation and core-fucosylation. ► Identification of aberrant N-glycosites with both CID and ETD. ► Quantification of N-glycosite aberrancy with isotope dimethyl labeling.
Co-reporter:Zhenbin Zhang, Fangjun Wang, Bo Xu, Hongqiang Qin, Mingliang Ye, Hanfa Zou
Journal of Chromatography A 2012 Volume 1256() pp:136-143
Publication Date(Web):21 September 2012
DOI:10.1016/j.chroma.2012.07.071
Strong cation exchange (SCX) chromatography is one of the most important separation modes in liquid chromatography and SCX column is widely applied in high resolution separation or fractionation of various samples. In this work, a sulfonate SCX hybrid monolithic column was successfully prepared by “one-pot” strategy and the hybrid monolith is well optimized to obtain homogenous structure. It was observed that this sulfonate SCX hybrid monolithic column had ∼7 times permeability (in water) and ∼3 times sample loading capacity (tested by dipeptide Gly-Tyr) comparing to particulate SCX column packed with commercial available material. Then, it was used as trap column for fast sample loading of the enriched phosphopeptides. Comparing to phosphate SCX polymer monolithic column, the number of identified phosphopeptides increased ∼19% due to the sulfonate group has higher retention strength than phosphate group for peptide cations. And the coverage of phosphoproteome obtained by sulfonate SCX hybrid monolithic column is similar to particulate packed SCX column, because they had identical sulfonate group to retain the peptide cations. Finally, the sulfonate SCX hybrid monolithic column was used as enzyme reactor for online protein digestion. Comparing to particulate SCX packed column, the identified peptides number increased 40% and the protein coverage increased 10%. This might be ascribed to the high porous structure and relative high surface area that elevated the digestion efficiency.Highlights► A sulfonate-silica hybrid SCX monolithic column was prepared by “one-pot” strategy. ► The SCX monolithic column had ∼7 times permeability (in water) and ∼3 times sample loading capacity (tested by dipeptide Gly-Tyr) comparing to packed SCX column. ► The SCX monolithic column was used as trap column for phosphoproteome analysis and as enzyme reactor for automated and rapid protein digestion.
Co-reporter:Liang Zhao, Hongqiang Qin, Ren’an Wu, Hanfa Zou
Journal of Chromatography A 2012 Volume 1228() pp:193-204
Publication Date(Web):9 March 2012
DOI:10.1016/j.chroma.2011.09.051
Sample preparation has been playing an important role in the analysis of complex samples. Mesoporous materials as the promising adsorbents have gained increasing research interest in sample preparation due to their desirable characteristics of high surface area, large pore volume, tunable mesoporous channels with well defined pore-size distribution, controllable wall composition, as well as modifiable surface properties. The aim of this paper is to review the recent advances of mesoporous materials in sample preparation with emphases on extraction of metal ions, adsorption of organic compounds, size selective enrichment of peptides/proteins, specific capture of post-translational peptides/proteins and enzymatic reactor for protein digestion.
Co-reporter:Zhenbin Zhang, Hui Lin, Junjie Ou, Hongqiang Qin, Ren’an Wu, Jing Dong, Hanfa Zou
Journal of Chromatography A 2012 Volume 1228() pp:263-269
Publication Date(Web):9 March 2012
DOI:10.1016/j.chroma.2011.07.048
A phenyl-silica hybrid monolithic column for capillary liquid chromatography (cLC) was prepared through “one-pot” process by con-currently using benzyl methacrylate and alkoxysilanes. The effects of the molar ratio of tetramethoxysilane/vinyltrimethoxysilane (TMOS/VTMS), polycondensation temperature, content of supramolecule template (cetyltrimethylammonium bromide, CTAB), ratio of N,N′-dimethylformamide/methanol (v/v), the volume of benzyl methacrylate on the morphologies of the prepared phenyl-silica hybrid monolithic columns were investigated in detail. The permeability of the hybrid monolithic column was calculated as 3.23 × 10−13 m2, and the minimum plate height was determined as 8.38 μm which corresponding to 119,000 theoretical plates per meter. Separation of various neutral, polar and basic analytes as well as small peptides on the hybrid monolithic column was achieved by cLC and showed high efficiency and satisfactory reproducibility. Moreover, the prepared hybrid monolithic column was also applied for the analysis of tryptic digests of bovine serum albumin (BSA), ovalbumin, α-casein, cytochrome C and myoglobin by cLC tandem mass spectrometry (cLC–MS/MS), and the results showed that the separation performance was close to that of the octadecylsilane (C18) packed capillary column which demonstrating its potential in proteome analysis. Moreover, since the prepolymerization system was mainly consisted of organic solvents (methanol and N,N′-dimethylformamide), various hydrophobic monomers could be potentially used to prepare organic-silica hybrid monolithic columns through “one-pot” approach.
Co-reporter:Junjie Ou, Hui Lin, Shouwan Tang, Zhenbin Zhang, Jing Dong, Hanfa Zou
Journal of Chromatography A 2012 Volume 1269() pp:372-378
Publication Date(Web):21 December 2012
DOI:10.1016/j.chroma.2012.09.022
A hybrid monolithic capillary column synthesized with (3-chloropropyl)-trimethoxysilane (CPTMS) and tetramethoxysilane (TMOS) via sol–gel chemistry was in situ coated with cellulose tris(3,5-dimethylphenyl-carbamate) (CDMPC) for enantioseparations in capillary electrochromatography (CEC) and capillary liquid chromatography (CLC). Prior to coating, the prepared CP-silica hybrid monolith was straightforwardly modified with diethylenetriamine (DETA) to introduce NH2 functionalities via the nucleophilic substitution reaction, which generate the stronger EOF for CEC. The coating condition was optimized to obtain a stable and reproducible chiral stationary phase for enantioseparation. The results indicated that racemic benzoin was baseline separated on the resulting hybrid monolith coated with 30 mg/mL CDMPC in CEC, while several racemates were successfully enantioseparated on the resulting CP-silica hybrid monolithic column coated with 60 mg/mL CDMPC in CLC with RP and NP modes.Highlights► A hybrid monolith was synthesized with CPTMS and TMOS via sol–gel chemistry. ► The hybrid monolith was straightforwardly modified to introduce NH2 functionality. ► Cellulose deriviative was coated on monolith for enantioseparations in CEC and CLC.
Co-reporter:Xue Xu, Ruibin Li, Ming Ma, Xia Wang, Yonghua Wang and Hanfa Zou
Soft Matter 2012 vol. 8(Issue 10) pp:2915-2923
Publication Date(Web):31 Jan 2012
DOI:10.1039/C2SM06811G
Organisms have evolved stress-inducible defense responses such as the P-glycoprotein (P-gp)-mediated efflux system to maintain chemical homeostasis in cells for both endogenous and xenobiotic compounds. However, despite the extensive focus on the potential interactions of P-gp with small molecules, the effect of nanoparticles on this transporter is scarcely reported. Thus in this work, in vitro experiments combined with molecular dynamics (MD) simulations were carried out to investigate the interactions of the multidrug resistance (MDR) protein P-gp with fullerene (C60), one of the most important nano-drug carriers. Upon exposure to fluorescence-labeledC60 (0–70 μg mL−1) for 2 h, significant accumulation of C60 is found in both the K562S and K562R cells, suggesting the incapability of P-gp to induce the efflux of this nanoparticle. In addition, in vitro inhibition assays also reveal that C60 does not obviously hinder P-gp-mediated rhodamine-123 transport in both K562S and K562R cells. The theoretical simulations further reveal the mechanism involved in C60-P-gp interactions, i.e., the binding of C60 barely induces the conformational changes of P-gp with RMSD of ∼4.8 Å and radius of gyration of ∼41.5 Å, and also no theoretical evidence shows that the C60 acts as a substrate or inhibitor of P-glycoprotein. These results demonstrate the potential of C60 as a good carrier candidate for MDR-targeted drug delivery, since organisms probably have not evolved to recognize this nanoparticle.
Co-reporter:Xu Wang, Yangyang Bian, Kai Cheng, Hanfa Zou, Samuel Sai-Ming Sun, and Jun-Xian He
Journal of Proteome Research 2012 Volume 11(Issue 4) pp:2301-2315
Publication Date(Web):2017-2-22
DOI:10.1021/pr2010764
Nitrogen (N) is an important nutrient and signal for plant growth and development. However, to date, our knowledge of how plants sense and transduce the N signals is very limited. To better understand the molecular mechanisms of plant N responses, we took two-dimensional gel-based proteomic and phosphoproteomic approaches to profile the proteins with abundance and phosphorylation state changes during nitrate deprivation and recovery in the model plant Arabidopsis thaliana. After 7-day-old seedlings were N-deprived for up to 48 h followed by 24 h recovery, a total of 170 and 38 proteins were identified with significant changes in abundance and phosphorylation state, respectively. Bioinformatic analyses implicate these proteins in diverse cellular processes including N and protein metabolisms, photosynthesis, cytoskeleton, redox homeostasis, and signal transduction. Functional studies of the selected nitrate-responsive proteins indicate that the proteasome regulatory subunit RPT5a and the cytoskeleton protein Tubulin alpha-6 (TUA6) play important roles in plant nitrate responses by regulating plant N use efficiency (NUE) and low nitrate-induced anthocyanin biosynthesis, respectively. In conclusion, our study provides novel insights into plant responses to nitrate at the proteome level, which are expected to be highly useful for dissecting the N response pathways in higher plants and for improving plant NUE.
Co-reporter:Yangyang Bian, Mingliang Ye, Chunxia Song, Kai Cheng, Chunli Wang, Xiaoluan Wei, Jun Zhu, Rui Chen, Fangjun Wang, and Hanfa Zou
Journal of Proteome Research 2012 Volume 11(Issue 5) pp:2828-2837
Publication Date(Web):2017-2-22
DOI:10.1021/pr300242w
Complete coverage of all phosphorylation sites in a proteome is the ultimate goal for large-scale phosphoproteome analysis. However, only making use of one protease trypsin for protein digestion cannot cover all phosphorylation sites, because not all tryptic phosphopeptides are detectable in MS. To further increase the phosphoproteomics coverage of HeLa cells, we proposed a tandem digestion approach by using two different proteases. By combining the data set of the first Glu-C digestion and the second trypsin digestion, the tandem digestion approach resulted in the identification of 8062 unique phosphopeptides and 8507 phosphorylation sites in HeLa cells. The conventional trypsin digestion approach resulted in the identification of 3891 unique phosphopeptides and 4647 phosphorylation sites. It was found that the phosphorylation sites identified from the above two approaches were highly complementary. By combining above two data sets, in total we identified 10899 unique phosphopeptides and 11262 phosphorylation sites, corresponding to 3437 unique phosphoproteins with FDR < 1% at peptide level. We also compared the kinase motifs extracted from trypsin, Glu-C, or a second trypsin digestion data sets. It was observed that basophilic motifs were more frequently found in the trypsin and the second trypsin digestion data sets, and the acidic motifs were more frequently found in the Glu-C digestion data set. These results demonstrated that our tandem digestion approach is a good complement to the conventional trypsin digestion approach for improving the phosphoproteomics analysis coverage of HeLa cells.
Co-reporter:Mingming Dong, Mingliang Ye, Kai Cheng, Chunxia Song, Yanbo Pan, Chunli Wang, Yangyang Bian, and Hanfa Zou
Journal of Proteome Research 2012 Volume 11(Issue 9) pp:4673-4681
Publication Date(Web):2017-2-22
DOI:10.1021/pr300503z
The Ser/Thr protein kinases fall into three major subgroups, pro-directed, basophilic, and acidophilic, on the basis of the types of substrate sequences that they preferred. Despite many phosphoproteomics efforts that have been taken for global profiling of phosphopeptides, methodologies focusing on analyzing a particular type of kinase substrates have seldom been reported. Selective enrichment of phosphopeptides from basophilic kinase substrates is difficult because basophilic motifs are cleaved by trypsin during digestion. In this study, we develop a negative enrichment strategy to enhance the identification of basophilic kinase substrates. This method is based on an observation that high pH strong anion exchange (SAX) chromatography can separate tryptic phosphopeptides according to the number of acidic amino acidic residues that they have. Thus, SAX was applied to deplete acidic phosphopeptides from the phosphopeptide mixture, which improved the coverage for the detection of basophilic kinase substrates. The SAX depletion approach was further combined with online SCX-RP separation for large-scale analysis of mouse liver phosphoproteome, which resulted in the identification of 6944 phosphorylated sites. It was found that motifs associated with basophilic kinases prevail for these identified phosphorylated sites.
Co-reporter:Fangjun Wang;Jun Zhu;Lianghai Hu;Hongqiang Qin;Mingliang Ye
Protein & Cell 2012 Volume 3( Issue 9) pp:669-674
Publication Date(Web):2012 September
DOI:10.1007/s13238-012-2934-4
The human serum proteome is closely associated with the state of the body. Endogenous peptides derived from proteolytic enzymes cleaving on serum proteins are widely studied due to their potential application in disease-specific marker discovery. However, the reproducibility of peptidome analysis of endogenous peptides is significantly influenced by the proteolytic enzymes within body fluids, thereby limiting the clinical use of the endogenous peptides. We comprehensively investigated the N and C terminus of endogenous peptides using peptidomics. The cleavage site patterns of the N and C terminus and adjacent sites from all the identified endogenous peptides were highly conserved under different sample preparation conditions, including long-term incubation at 37°C and pretreatment with repeated freeze-thaw cycles. Furthermore, a distinguishable cleavage site pattern was obtained when a different disease serum was analyzed. The conserved cleavage site pattern derived from proteolytic enzymes holds potential in highly specific disease diagnosis.
Co-reporter:Zhenbin Zhang, Minghuo Wu, Ren’an Wu, Jing Dong, Junjie Ou, and Hanfa Zou
Analytical Chemistry 2011 Volume 83(Issue 9) pp:3616
Publication Date(Web):April 1, 2011
DOI:10.1021/ac200414r
Perphenylcarbamoylated β-cyclodextrin-silica (Ph-β-CD-silica) hybrid monolithic columns for enantioseparation in capillary liquid chromatography (cLC) have been prepared by a “one-pot” approach via the polycondensation of alkoxysilanes and in situ copolymerization of mono (6A-N-allylamino-6A-deoxy)-Ph-β-CD and vinyl group on the precondensed siloxanes. The morphologies of the Ph-β-CD-silica hybrid monolithic columns were characterized by optical microscopy and scanning electron microscopy (SEM), showing the uniform monolithic matrixes tightly bonded onto the capillary wall. The content of Ph-β-CD incorporated in monolithic matrix by the “one-pot” approach was ca. 2.9 times higher than that by postmodification method. The permeability of the Ph-β-CD-silica chiral hybrid monolithic column was 3.63 × 10−14 m2, and the minimum plate height was 12 μm corresponding to 83 300 theoretical plates/meter. Enantioseparations of 13 racemates were achieved by the Ph-β-CD-silica hybrid monolithic column. In this work, since the prepolymerization system mainly consisted of organic solvent (methanol (MeOH), N,N-dimethylformamide (DMF)), the limitation and difficulty of the use of water insoluble organic monomers in the previously reported “one-pot” method was circumvented. Therefore, various β-CD derivatives as well as other hydrophobic monomers could thus be used to prepare organic−silica hybrid monolithic columns with the “one-pot” process.
Co-reporter:Chunxia Song, Fangjun Wang, Mingliang Ye, Kai Cheng, Rui Chen, Jun Zhu, Yexiong Tan, Hongyang Wang, Daniel Figeys, and Hanfa Zou
Analytical Chemistry 2011 Volume 83(Issue 20) pp:7755
Publication Date(Web):September 8, 2011
DOI:10.1021/ac201299j
Accurately quantifying the changes of phosphorylation level on specific sites is crucial to understand the role of protein phosphorylation in physiological and pathological processes. Here, a pseudo triplex stable isotope dimethyl labeling approach was developed to improve the accuracy and the throughput of comprehensive quantitative phosphoproteome analyses. In this strategy, two identical samples are labeled with light and heavy isotopes, respectively, while another comparative sample is labeled with an intermediate isotope. Two replicated quantification results were achieved in just one experiment, and the relative standard deviation (RSD) criterion was used to control the quantification accuracy. Compared with the conventional duplex labeling approach, the number of quantified phosphopeptides increased nearly 50% and the experimental time was reduced by 50% under the same quantification accuracy. Combined with the automated online reversed phase-strong cation exchange-reversed phase (RP-SCX-RP) multidimensional separation system, a comparative phosphoproteome analysis of hepatocellular carcinoma (HCC) and normal human liver tissues was performed. Over 1800 phosphopeptides corresponding to ∼2000 phosphorylation sites were quantified reliably in a 42 h multidimensional analysis. The pro-directed motifs, which were mainly associated with the extracellular signal-regulated kinases (ERKs), were observed as being overrepresented in the regulated phosphorylation sites, and some quantification results of phosphorylation sites were validated by the other studies. Therefore, this pseudo triplex labeling approach was demonstrated as a promising alternative for the comprehensive quantitative phosphoproteome analysis.
Co-reporter:Hongqiang Qin, Liang Zhao, Ruibin Li, Ren’an Wu, and Hanfa Zou
Analytical Chemistry 2011 Volume 83(Issue 20) pp:7721
Publication Date(Web):September 7, 2011
DOI:10.1021/ac201198q
Many diseases are characterized by the changes of either glycan structure or glycosylation site of glycoproteins. The glycan profiling can provide the overview of glycosylation in despite of the absence of the glycosylation sites, which in turn simplifies the complexity of disease diagnosis. Herein, we describe a simple method to profile the N-linked glycans by MALDI-TOF MS with the enrichment using oxidized ordered mesoporous carbon, taking advantages of the size-exclusive effect of mesopore against proteins as well as the interaction between glycans and carbon. Twenty four N-linked glycans derived from ovalbumin could be efficiently detected with high signal-to-noise (S/N) ratios and sufficient peak intensities. In the analysis of complex serum samples, 32 N-linked glycans could be profiled, and 5 (4 core-fucosylated glycans) of them were distinguished from liver cancer and healthy samples.
Co-reporter:Fangjun Wang, Chunxia Song, Kai Cheng, Xinning Jiang, Mingliang Ye, and Hanfa Zou
Analytical Chemistry 2011 Volume 83(Issue 21) pp:8078
Publication Date(Web):September 19, 2011
DOI:10.1021/ac201833j
Protein phosphorylation is a ubiquitous post-translational modification that regulates almost all cellular processes. The analysis of protein phosphorylation is challenging due to the high dynamic range and low abundance natures of the analyte. Shotgun based proteomics has emerged as a very useful platform to achieve a comprehensive phosphoproteome analysis in considerable depth. In the past few years, significant breakthroughs on the large scale phosphorylation analysis have been witnessed along with the great development of related technologies. The combination of effective enrichment materials, refined analysis workflows, new type of powerful mass spectrometers, and sophisticated bioinformatic tools greatly boost the performance of comprehensive phosphoproteome analysis. In this Perspective, we briefly reviewed recent technological developments on the enrichment materials, prefractionation workflows, and different mass spectrometry fragmentation modes as well as software tools for phosphoproteome identification and quantification. Then, we described the current challenges and potential directions for the future of comprehensive phosphoproteome analysis. We also provide perspectives on how to further improve the performance of related analysis methods and technologies.
Co-reporter:Yingzhuang Chen, Minghuo Wu, Keyi Wang, Bo Chen, Shouzhuo Yao, Hanfa Zou, Lihua Nie
Journal of Chromatography A 2011 Volume 1218(Issue 44) pp:7982-7988
Publication Date(Web):4 November 2011
DOI:10.1016/j.chroma.2011.09.002
A novel thiol-ene “click” strategy for the preparation of monolithic trypsin microreactor was proposed. The hybrid organic–inorganic monolithic capillary column with ene-functionality was fabricated by sol–gel process using tetramethoxysilane (TMOS) and γ-methacryloxypropyltrimethoxysilane (γ-MAPS) as precursors. The disulfide bonds of trypsin were reduced to form free thiol groups. Then the trypsin containing free thiol groups was attached on the γ-MAPS hybrid monolithic column with ene-functionality via thiol-ene click chemistry to form a trypsin microreactor. The activity of the trypsin microreactor was characterized by detecting the substrate (Nα-p-tosyl-l-arginine methyl ester hydrochloride, TAME) and the product (Nα-p-tosyl-l-arginine, TA) with on-line capillary zone electrophoresis. After investigating various synthesizing conditions, it was found that the microreactor with poly(N,N′-methylenebisacrylamide) as spacer can deliver the highest activity, yielding a rapid reaction rate. After repeatedly sampling and analyzing for 100 times, the monolithic trypsin microreactor still remained 87.5% of its initial activity. It was demonstrated that thiol-ene “click” strategy for the construction of enzyme microreactor is a promising method for the highly selective immobilization of proteins under mild conditions, especially enzymes with free thiol radicals.Highlights► Vinyl silica hybrid monolith was fabricated. ► Monolithic based microreactor was prepared via thiol-ene “click” strategy. ► Poly(N,N′-methylenebisacrylamide) as spacer can deliver the highest activity. ► Thiol-ene “click” strategy is a promising method for the immobilization of proteins with free thiol radicals under mild conditions.
Co-reporter:Hongqiang Qin;Peng Gao;Fangjun Wang;Liang Zhao;Jun Zhu;Dr. Aiqin Wang;Dr. Tao Zhang;Dr. Ren'an Wu;Dr. Hanfa Zou
Angewandte Chemie International Edition 2011 Volume 50( Issue 51) pp:12218-12221
Publication Date(Web):
DOI:10.1002/anie.201103666
Co-reporter:Yongkang Jiang, Hongwei Liu, Hong Li, Fangjun Wang, Kai Cheng, Guangdong Zhou, Wenjie Zhang, Mingliang Ye, Yinlin Cao, Wei Liu, Hanfa Zou
Biomaterials 2011 32(17) pp: 4085-4095
Publication Date(Web):
DOI:10.1016/j.biomaterials.2011.02.033
Co-reporter:Hongqiang Qin;Peng Gao;Fangjun Wang;Liang Zhao;Jun Zhu;Dr. Aiqin Wang;Dr. Tao Zhang;Dr. Ren'an Wu;Dr. Hanfa Zou
Angewandte Chemie 2011 Volume 123( Issue 51) pp:12426-12429
Publication Date(Web):
DOI:10.1002/ange.201103666
Co-reporter:Yu Zhang, Chen Chen, Hongqiang Qin, Ren'an Wu and Hanfa Zou
Chemical Communications 2010 vol. 46(Issue 13) pp:2271-2273
Publication Date(Web):21 Jan 2010
DOI:10.1039/B921331G
Ti-hexagonal mesoporous silica (Ti-HMS) with high titanium content has been synthesized. The Ti-HMS has been applied as a potential adsorbent for the selective capture of phosphopeptides, due to the strong affinity interaction between the incorporated titanium in the framework and the phosphoryl groups of phosphopeptides.
Co-reporter:Qianhao Min, Ren'an Wu, Liang Zhao, Hongqiang Qin, Mingliang Ye, Jun-Jie Zhu and Hanfa Zou
Chemical Communications 2010 vol. 46(Issue 33) pp:6144-6146
Publication Date(Web):27 Jul 2010
DOI:10.1039/C0CC00619J
In this study, the concept of size-selective proteolysis was first described by using the mesoporous silica-based trypsin nanoreactor. For analysis of a complex protein sample, low-MW proteins were preferentially digested for identification while high-MW proteins were excluded from digestion.
Co-reporter:Chunxia Song, Mingliang Ye, Guanghui Han, Xinning Jiang, Fangjun Wang, Zhiyuan Yu, Rui Chen and Hanfa Zou
Analytical Chemistry 2010 Volume 82(Issue 1) pp:53
Publication Date(Web):December 1, 2009
DOI:10.1021/ac9023044
Protein phosphorylation regulates a series of important biological processes in eukaryotes. However, the phosphorylation sites found up to now are far below than that actually exists in proteins due to the extreme complexity of the proteome sample. Here a new reversed-phase-reversed-phase liquid chromatography (RP-RPLC) approach was developed for multidimensional separation of phosphopeptides. In this approach, a large number of fractions were collected from the first dimensional RPLC separation at high pH. And then these fractions were pooled every two fractions with equal time interval, one from the early eluted section and another one from the later eluted section. The pooled fractions were finally submitted to RPLC−tandem mass spectrometry (MS/MS) analysis at low pH. It was found the resulting 2D separation was highly orthogonal and yielded more than 30% phosphopeptide identifications over the conventional RP-RPLC approach. This study provides a powerful approach for efficient separation of phosphopeptides and global phosphorylation analysis, where the orthogonality of 2D separation is greatly improved and the first dimensional separation is of high resolution.
Co-reporter:Fangjun Wang, Rui Chen, Jun Zhu, Deguang Sun, Chunxia Song, Yifeng Wu, Mingliang Ye, Liming Wang and Hanfa Zou
Analytical Chemistry 2010 Volume 82(Issue 7) pp:3007
Publication Date(Web):March 15, 2010
DOI:10.1021/ac100075y
Multidimensional separation is often applied for large-scale qualitative and quantitative proteome analysis. A fully automated system with integration of a reversed phase-strong cation exchange (RP-SCX) biphasic trap column into vented sample injection system was developed to realize online sample loading, isotope dimethyl labeling and online multidimensional separation of the proteome samples. Comparing to conventionally manual isotope labeling and off-line fractionation technologies, this system is fully automated and time-saving, which is benefit for improving the quantification reproducibility and accuracy. As phosphate SCX monolith was integrated into the biphasic trap column, high sample injection flow rate and high-resolution stepwise fractionation could be easily achieved. ∼ 1000 proteins could be quantified in ∼30 h proteome analysis, and the proteome coverage of quantitative analysis can be further greatly improved by prolong the multidimensional separation time. This system was applied to analyze the different protein expression level of HCC and normal human liver tissues. After three times replicated analysis, finally 94 up-regulated and 249 down-regulated (HCC/Normal) proteins were successfully obtained. These significantly regulated proteins are widely validated by both gene and proteins expression studies previously. Such as some enzymes involved in urea cycle, methylation cycle and fatty acids catabolism in liver were all observed down-regulated.
Co-reporter:Minghuo Wu, Ren’an Wu, Ruibing Li, Hongqiang Qin, Jing Dong, Zhenbin Zhang and Hanfa Zou
Analytical Chemistry 2010 Volume 82(Issue 13) pp:5447
Publication Date(Web):May 28, 2010
DOI:10.1021/ac1003147
An inorganic−organic hybrid monolithic capillary column was synthesized via thermal free radical copolymerization within the confines of a capillary using a polyhedral oligomeric silsesquioxane (POSS) reagent as the inorganic−organic hybrid cross-linker and a synthesized long carbon chain quaternary ammonium methacrylate of N-(2-(methacryloyloxy)ethyl)-dimethyloctadecylammonium bromide (MDOAB) as the organic monomer. The preparation process was as simple as pure organic polymer-based monolithic columns instead of using POSS as the nanosized inorganic−organic hybrid blocks (cross-linker) of the monolithic matrix. The pore properties and permeability could be tuned by the composition of the polymerization mixture. The characterization and evaluation results indicated that the synthesized MDOA−POSS hybrid monolith possessed the merits of organic polymer-based monoliths and silica-based monoliths with good mechanical and pH (pH 1−11) stabilities, which may be attributed to the incorporation of the rigid nanosized silica core of POSS. Column efficiencies of 223 000 and 50 000 N/m were observed in capillary electro-driven chromatography (CEC) and μ-HPLC, respectively. Peptides and standard proteins were baseline separated by this hybrid column in CEC and μ-HPLC, respectively, as well. The separation of bovine serum albumin (BSA) tryptic digest was also attempted to show its potential application in proteome analysis.
Co-reporter:Xinning Jiang, Mingliang Ye, Guanghui Han, Xiaoli Dong and Hanfa Zou
Analytical Chemistry 2010 Volume 82(Issue 14) pp:6168
Publication Date(Web):June 22, 2010
DOI:10.1021/ac100975t
Data dependent neutral loss triggered MS3 methodology (NLMS3) is often applied to acquire MS data for the analysis of phosphopeptides. Some phosphopeptides tend to seriously lose the phosphate and result in MS2 spectra with poor fragments and fragment-rich MS3 spectra, while some phosphopeptides do not lose phosphate and result in nice MS2 spectra. Since different phosphopeptides have fragment spectra with different characteristics, filtering all of the phosphopeptide identifications by setting a global filter criteria may be inappropriate and result in low sensitivity. In this study, we developed a classification filtering strategy to improve the phosphopeptide identification and phosphorylation site localization. Phosphopeptide identifications were classified into four classes according to their different characteristics, and then, the identifications from each class of mass spectra were processed and filtered separately using different filtering strategies. It was found that the overlap of phosphopeptide identifications from different classes was low and the classification strategy significantly improved the coverage of the phosphoproteome analysis. Compared with MS2 strategy and multiple stage activation (MSA) strategy, NLMS3 with the classification filtering strategy was demonstrated to have higher sensitivity and higher performance in localizing the phosphorylation to specific sites.
Co-reporter:Minghuo Wu, Yingzhuang Chen, Ren’an Wu, Ruibing Li, Hanfa Zou, Bo Chen, Shouzhuo Yao
Journal of Chromatography A 2010 Volume 1217(Issue 26) pp:4389-4394
Publication Date(Web):25 June 2010
DOI:10.1016/j.chroma.2010.03.019
A chloropropyl-functionalized silica (CP-silica) hybrid monolithic column was synthesized within the confines of a capillary via the sol–gel process using tetramethoxysilane (TMOS) and (3-chloropropyl)-trimethoxysilane (CPTMS) as the precursors. The resulting CP-silica hybrid monolith inside the capillary showed homogeneous macroporous morphology and was well attached to the inner wall of the capillary. The obtained CP-silica hybrid monolithic capillary column demonstrated the inherent hydrophobic property and could be applied as a reversed-phase stationary phase in CEC directly. Due to the great chemical reactivity of the incorporated chloro groups on the hybrid silica monolithic matrix, the chloropropyl moieties on the surface of the hybrid silica monolith matrix could be conveniently further modified by a tertiary amine of N,N-dimethyl-N-dodecylamine (DMDA) via the nucleophilic substitution reaction at 70 °C to introduce a dodecyl groups (C12) onto the CP-silica hybrid monolithic matrix. The resulting C12-silica hybrid monolithic column not only demonstrated the significantly enhanced hydrophobic property in the separation of alkylbenzenes in reversed-phase capillary electrochromatography (RP-CEC), but also the strong electroosmotic flow (EOF) in a wide pH range. Five alkylbenzenes could be baseline separated in 3 min with column efficiency ranging from 189 700 to 221 000 N/m with a 70% ACN running buffer in CEC.
Co-reporter:Chen Wang, Jiangrui Zhou, Shuowen Wang, Mingliang Ye, Chunlei Jiang, Guorong Fan and Hanfa Zou
Journal of Proteome Research 2010 Volume 9(Issue 6) pp:3225-3234
Publication Date(Web):2017-2-22
DOI:10.1021/pr1001274
This study investigated the mechanisms involved in the antinociceptive action induced by levo-tetrahydropalmatine (l-THP) in the formalin test by combined comparative and chemical proteomics. Rats were pretreated with l-THP by the oral route (40 mg/kg) 1 h before formalin injection. The antinociceptive effect of l-THP was shown in the first and second phases of the formalin test. To address the mechanisms by which l-THP inhibits formalin-induced nociception in rats, the combined comparative and chemical proteomics were applied. A novel high-throughput comparative proteomic approach based on 2D-nano-LC−MS/MS was applied to simultaneously evaluate the deregulated proteins involved in the response of l-THP treatment in formalin-induced pain rats. Thousands of proteins were identified, among which 17 proteins survived the stringent filter criteria and were further included for functional discussion. Two proteins (Neurabin-1 and Calcium-dependent secretion activator 1) were randomly selected, and their expression levels were further confirmed by Western Blots. The results matched well with those of proteomics. In the present study, we also described the development and application of l-THP immobilized beads to bind the targets. Following incubation with cellular lysates, the proteome interacting with the fixed l-THP was identified. The results of comparative and chemical proteomics were quite complementary. Although the precise roles of these identified moleculars in l-THP-induced antinociception need further study, the combined results indicated that proteins associated with signal transduction, vesicular trafficking and neurotransmitter release, energy metabolism, and ion transport play important roles in l-THP-induced antinociception in the formalin test.
Co-reporter:Houjiang Zhou, Fred Elisma, Nicholas J. Denis, Theodore G. Wright, Ruijun Tian, Hu Zhou, Weimin Hou, Hanfa Zou and Daniel Figeys
Journal of Proteome Research 2010 Volume 9(Issue 3) pp:1279-1288
Publication Date(Web):2017-2-22
DOI:10.1021/pr900767j
Protein phosphorylation is an important post-translational modification involved in the regulation of many cellular processes. Mass spectrometry has been successfully used to identify protein phosphorylation in specific pathways and for global phosphoproteomic analysis. However, phosphoproteomics approaches do not evaluate the subcellular localization of the phosphorylated forms of proteins, which is an important factor for understanding the roles of protein phosphorylation on a global scale. The in-depth mapping of protein phosphorylation at the subcellular level necessitates the development of new methods capable of specifically and efficiently enriching phosphopeptides from highly complex samples. Here, we report a novel microfluidic device called the phosphoproteomic reactor that combines efficient processing of proteins followed by phosphopeptide enrichment by Ti-IMAC. To illustrate the potential of this novel technology, we mapped the phosphoproteins in subcellular organelles of liver cells. Fifteen subcellular fractions from liver cell cultures were processed on the phosphoproteomic reactor in combination with nano-LC-MS/MS analysis. We identified thousands of phosphorylation sites in over 600 phosphoproteins in different organelles using minute amounts of starting material. Overall, this approach provides a new avenue for studying the phosphoproteome of the subcellular organelles.
Co-reporter:Fangjun Wang;Guanghui Han;Zhiyuan Yu;Xinning Jiang;Shutao Sun;Rui Chen;Mingliang Ye
Journal of Separation Science 2010 Volume 33( Issue 13) pp:1879-1887
Publication Date(Web):
DOI:10.1002/jssc.200900718
Abstract
It is one of the key issues to develop powerful fractionating method to increase the identification of the low-abundance phosphopeptides. In this study, a semi-online 2-D LC separation strategy based on three-step fractionation of the enriched peptides on strong anion-exchange trap column was developed. It was demonstrated that the sensitivity and phosphoproteome coverage obtained by this fractionating method with strong anion-exchange trap column is much higher than those by the conventional methods based on C18 trap column. In addition, when the same amount of sample was loaded, the number of identified phosphopeptides had increased 108%. Combination of this three-step fractionation method with RPLC-MS/MS analysis by 300 min RP-gradient separation was applied to phosphoproteome analysis of human liver proteins, and 853 unique phosphopeptides was positively identified from 500 μg tryptic digest of human liver proteins. After three cycles' consecutive analyses, 1554 unique phosphopeptides and 1566 phosphorylated sites were totally identified from 735 phosphorylated proteins at a false discovery rate of <1% in about 54 h of analysis time.
Co-reporter:Xiaoyan Liu;Chunxia Song;Rui Chen;Xinning Jiang;Yan Jin
Chinese Journal of Chemistry 2010 Volume 28( Issue 9) pp:1665-1672
Publication Date(Web):
DOI:10.1002/cjoc.201090282
Abstract
Proteomics is a rapidly emerging set of key technologies that are of major importance for proteins and drug development process, especially when mass spectrometry (MS) is being used for high-throughput characterization and identification of proteins. Since the safer and healthier angiotensin I-converting enzyme (ACE) inhibitors are extremely concerned, many research groups have combed for novel ACE inhibitors from food components by different approaches. Here, shotgun proteomics technology aided with structure-activity analysis was applied to screen ACE inhibitory peptides from hydrolyzed red deer plasma. The peptides were analysed by mass spectrometry after primary separation with Sephadex G-25 chromatography. 36 peptides were identified by searching red deer database and 165 peptide sequences derived were identified in mammalian database. Amino acid sequences of peptide and bioactivity relationship have been developed as a faster and useful way to predict and screen new inhibitors. Depending on the relationship of peptide structure and ACE inhibitory activity, a nonapeptide, VYNEGLPAP, was predicted with ACE inhibitory activity. The activity was verified by synthesized VYNEGLPAP and the 50% inhibition concentration (IC50) was 3.1 µmol·L−1. VYNEGLPAP had good thermal stability, pH stability and strong enzyme-resistant properties against gastrointestinal protease. Kinetic experiments demonstrated that inhibitory kinetic mechanism of this peptide was competitive. This study demonstrated the possibility of screening bioactive peptides from protein hydrolysates mixture based on shotgun proteomics technology, which will provide a potential convenient method to screening bioactive peptides from protein source.
Co-reporter:Zhiyuan Yu, Ren’an Wu, Minghuo Wu, Liang Zhao, Ruibin Li, Hanfa Zou
Colloids and Surfaces B: Biointerfaces 2010 Volume 78(Issue 2) pp:222-228
Publication Date(Web):1 July 2010
DOI:10.1016/j.colsurfb.2010.03.004
The level of serum free copper is greatly elevated in Wilson's disease. For patients with acute Wilson's disease, liver transplantation is the only lifesaving treatment. Plasma exchange or albumin dialysis is often used as a bridge to liver transplantation to maintain a stable clinical status for patients. Hemoperfusion is another effective therapy in removing toxins from the plasma. However, hemoperfusion has not been reported to remove copper due to lack of copper specific adsorbent. In this work, copper chelating agents, triethylenetetramine and tetraethylenepentamine, were covalently immobilized onto macroporous poly(glycidyl methacrylate-co-trimethylolpropane trimethacrylate) microspheres to prepare copper specific adsorbents. The resulting adsorbents demonstrated good adsorption capacities of 63.44 and 58.48 mg/g, respectively, for Cu2+ ion. Additionally, with the interference of other metal ions such as Fe2+, Mg2+, Zn2+ and Ca2+, the prepared copper adsorbents still demonstrated good specificity toward Cu2+ ion. These results indicate that the adsorbents are promising adsorbents in hemoperfusion therapy for selective removal of copper for patients with severe Wilson's disease.
Co-reporter:Chen Wang;Shuowen Wang;Guorong Fan
Analytical and Bioanalytical Chemistry 2010 Volume 396( Issue 5) pp:1731-1740
Publication Date(Web):2010 March
DOI:10.1007/s00216-009-3409-1
Formalin-induced pain models were used in rats to evaluate the antinociceptive effect of the total alkaloids of Corydalis yanhusuo (TAC). The results indicated that formalin-evoked spontaneous nociceptive responses (licking behavior) could be inhibited significantly by giving (intragingival) TAC at a single dose of 150 mg/kg. Subsequently, an online comprehensive two-dimensional biochromatography method with a silica-bonded human serum albumin (HSA) column in the first dimension and a monolithic ODS column in the second was developed. The absorbed bioactive components were screened by comparing and contrasting the components detected in the plasma and striatum with those in TAC. More than 100 compounds were separated and detected in the TAC, among which 13 compounds were identified. About 40 compounds (seven compounds identified) were absorbed into the plasma with appropriate concentrations and about 20 compounds (four compounds identified) passed through the blood-brain barrier into the striatum. Of interest, four compounds (protopine, glaucine, tetrahydropalmatine, and corydaline) which were reported to possess profound antinociceptive effects exhibited high concentrations in the striatum. This may result from their synergistic effects in regulating the formalin-induced nociception. The results indicated that the comprehensive two-dimensional biochromatography method developed is capable of screening the bioactive components in Corydalis yanhusuo and providing valuable information for understanding the mechanisms by which Corydalis yanhusuo alleviates nociception.
Co-reporter:Jun Zhu;FangJun Wang;XiaoLi Dong;MingLiang Ye
Science China Chemistry 2010 Volume 53( Issue 4) pp:759-767
Publication Date(Web):2010 April
DOI:10.1007/s11426-010-0127-7
Peptidomics draws more and more attention in discovering useful biomarkers for early diagnosis of disease. However, there is lack of efficient quantification strategy in peptidome analysis. In this study, a strategy with label-free quantification of the targeted endogenous peptides based on peak intensity using μUPLC-Q-TOF-MS/MS was developed for quantitative peptidome analysis of human serum. Different amounts of standard BSA tryptic digesting peptides were added into the same serum extracts for evaluation of the developed strategy, and it was observed that the average relative error of the targeted peptides was 6.42%, which was superior to the result obtained directly by commercially available software PLGS. It was also demonstrated that this quantification strategy could obviously increase the detection sensitivity of the peptide by DDA analysis. Then, this strategy was applied to comparatively analyze the peptides extracted from the serum of HCC or breast cancer patients and healthy individuals, respectively. Peptides with charge states up to 5 and molecular weight over 4000 can be reliably identified and quantified. This quantitative analysis method based on μUPLC-Q-TOF-MS/MS exhibited superior sensitivity than that by MALDI-TOF-MS commonly used in peptidome analysis. Finally, some interesting endogenous peptides related to corresponding diseases were successfully obtained.
Co-reporter:Ruibin Li, Ren’an Wu, Liang Zhao, Minghuo Wu, Ling Yang and Hanfa Zou
ACS Nano 2010 Volume 4(Issue 3) pp:1399
Publication Date(Web):February 11, 2010
DOI:10.1021/nn9011225
Multidrug resistance (MDR), which is related to cancer chemotherapy, tumor stem cells, and tumor metastasis, is a huge obstacle for the effective cancer therapy. One of the underlying mechanisms of MDR is the increased efflux of anticancer drugs by overexpressed P-glycoprotein (P-gp) of multidrug resistant cells. In this work, the antibody of P-gp (anti-P-gp) functionalized water-soluble single-walled carbon nanotubes (Ap-SWNTs) loaded with doxorubicin (Dox), Dox/Ap-SWNTs, were synthesized for challenging the MDR of K562 human leukemia cells. The resulting Ap-SWNTs could not only specifically recognize the multidrug resistant human leukemia cells (K562R), but also demonstrate the effective loading and controllable release performance for Dox toward the target K562R cells by exposing to near-infrared radiation (NIR). The recognition capability of Ap-SWNTs toward the K562R cells was confirmed by flow cytometry (FCM) and confocal laser scanning microscopy (CLSM). The binding affinity of Ap-SWNTs toward drug-resistant K562R cells was ca. 23-fold higher than that toward drug-sensitive K562S cells. Additionally, CLSM indicated that Ap-SWNTs could specifically localize on the cell membrane of K562R cells and the fluorescence of Dox in K562R cells could be significantly enhanced after the employment of Ap-SWNTs as carrier. Moreover, the composite of Dox and Ap-SWNTs (Dox/Ap-SWNTs) expressed 2.4-fold higher cytotoxicity and showed the significant cell proliferation suppression toward K562R leukemia cells (p < 0.05) as compared with free Dox which is popularly employed in clinic trials. These results suggest that the Ap-SWNTs are the promising drug delivery vehicle for overcoming the MDR induced by the overexpression of P-gp on cell membrane. Ap-SWNTs loaded with drug molecules could be used to suppress the proliferation of multidrug resistant cells, destroy the tumor stem cells, and inhibit the metastasis of tumor.Keywords: carbon nanotubes; doxorubicin; drug delivery; leukemia cell; multidrug resistance
Co-reporter:Lianghai Hu, Houjiang Zhou, Yinghua Li, Shutao Sun, Lihai Guo, Mingliang Ye, Xiaofeng Tian, Jianren Gu, Shengli Yang and Hanfa Zou
Analytical Chemistry 2009 Volume 81(Issue 1) pp:94
Publication Date(Web):December 2, 2008
DOI:10.1021/ac801974f
Phosphorylation is one of the most important post-translational modifications of proteins, which modulates a wide range of biological functions and activities of proteins. The phosphorylation of proteins is also associated with the pathway of cancer cells. We have previously enriched the low molecular weight proteome from human plasma based on the combination of size exclusion and adsorption mechanism by using highly ordered mesoporous silica particles. Herein, highly ordered mesoporous silica particles were modified with titanium phosphonate to selectively capture the phosphopeptides from complex peptide and protein mixtures. The limit of detection for phosphopeptides from β-casein and standard phosphopeptide spiked in human serum was as low as 1.25 fmol based on MALDI-TOFMS detection. The modified mesoporous silica particles were further used to enrich phosphopeptides from serum of hepatocellular carcinoma patients and healthy individuals and then analyzed with MALDI-TOFMS. The combination of isobaric tagging for relative and absolute quantitation labeling with MALDI-TOFMS/MS was further applied to validate the serum phosphopeptide profiling result of MALDI-TOFMS. The profiling of the serum phosphopeptides between the cancer patients and healthy persons was distinguishingly different, which indicated the potential ability of this technique for cancer diagnosis and biomarker discovery. The approach developed here would be applicable to other biological samples and a wide variety of diseases.
Co-reporter:Minghuo Wu, Ren’an Wu, Fangjun Wang, Lianbing Ren, Jing Dong, Zhen Liu and Hanfa Zou
Analytical Chemistry 2009 Volume 81(Issue 9) pp:3529
Publication Date(Web):April 1, 2009
DOI:10.1021/ac9000749
A “one-pot” process for the preparation of organic-silica hybrid capillary monolithic columns by concurrently using organic monomers and alkoxysilanes was described. In this process, the hydrolyzed alkoxysilanes of tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS) as precursors for the synthesis of a silica-based monolith using the sol−gel method and the organic monomer (allyldimethyldodecylammonium bromide (ADDAB) or acrylamide (AA)) with vinyl groups for free radical polymerization along with the initiator of azobisisobutyronitrile (AIBN) were concurrently introduced into a pretreated capillary; after that, the polycondensation of alkoxysilanes and the copolymerization of organic monomers and as-precondensed siloxanes were subsequently carried out within the confines of a capillary at the proper reaction conditions. Two types of organic-silica hybrid capillary monolithic columns with hydrophobic and hydrophilic properties have been fabricated, respectively, by this “one-pot” process using two different organic monomers of ADDAB and AA. The morphologies of the synthesized organic-silica hybrid monolithic columns were characterized by scanning electron microscopy (SEM). The performances of these organic-silica monolithic columns were investigated by capillary electrochromatography (CEC). The retention behaviors of the neutral and polar compounds on the resulting hydrophobic and hydrophilic organic-silica hybrid monolithic columns confirmed the successful incorporation of organic monomers in the silica monolithic matrix. In addition, the ADDA-silica hybrid capillary monolithic column was also applied in the analysis of tryptic digests of bovine serum albumin (BSA) and mouse liver extract by microliquid chromatography−tandem mass spectrometry (μLC−MS/MS) for demonstrating its potential in proteome analysis.
Co-reporter:Guanghui Han, Mingliang Ye, Xinning Jiang, Rui Chen, Jian Ren, Yu Xue, Fangjun Wang, Chunxia Song, Xuebiao Yao and Hanfa Zou
Analytical Chemistry 2009 Volume 81(Issue 14) pp:5794
Publication Date(Web):June 12, 2009
DOI:10.1021/ac900702g
Since the emergence of proteomics, much attention has been paid to the development of new technologies for phosphoproteomcis analysis. Compared with large scale phosphorylation analysis at the proteome level, comprehensive and reliable phosphorylation site mapping of individual phosphoprotein is equally important. Here, we present a modified target-decoy database search strategy for confident phosphorylation site analysis of individual phosphoproteins without manual interpretation of spectra. Instead of using all protein sequences in a proteome database of an organism for the construction of a target-decoy database for phosphoproteome analysis, the composite database constructed for phosphorylation site analysis of individual phosphoproteins only included the sequences of the individual target proteins and a decoy version of a small inhomogeneous protein database. It was found that the confidence of phosphopeptide identifications could be effectively controlled when the acquired MS2 and MS3 spectra were searched against the above composite database followed with data processing. Because of the small size of the composite database, the computation time for the database search is very short, which allows the adoption of low-specificity proteases for protein digestion to increase the coverage of phosphorylation site mapping. The sensitivity and comprehensive phosphorylation site mapping of this approach was demonstrated by using two standard phosphoprotein samples of α-casein and β-casein, and this approach was further applied to analyze the phosphorylation of the cyclic AMP-dependent protein kinase (PKA), which resulted in the identification of 17 phosphorylation sites, including five novel sites on four PKA subunits.
Co-reporter:Fangjun Wang, Jing Dong, Mingliang Ye, Ren’an Wu, Hanfa Zou
Analytica Chimica Acta 2009 Volume 652(1–2) pp:324-330
Publication Date(Web):12 October 2009
DOI:10.1016/j.aca.2009.06.066
Capillary column plays an important role in nano-flow liquid chromatography coupled with tandem mass spectrometry for dealing with the high dynamic range and complexity of protein samples in shotgun proteome analysis. In this study, the integrated monolithic frit into the particulate capillary (IMFPC) column was prepared. By comparing the prepared IMFPC column with conventionally fritless capillary column, smaller size of packing materials could be easily packed into the capillary to achieve higher average peak capacity and proteome coverage. As the monolithic emitter was integrated onto this type of column, the void volume between packing particles and electrospray emitter was eliminated and the electrospray quality was improved. The prepared IMFPC column was applied to proteome analysis of mouse liver extracts, and it was observed that the number of identified proteins and peptides increased 14.9 and 12.9% as well as the peak capacity increased 11.6% by using IMFPC column over conventionally fritless capillary column.
Co-reporter:Zhiyuan Yu, Guanghui Han, Shutao Sun, Xinning Jiang, Rui Chen, Fangjun Wang, Ren’an Wu, Mingliang Ye, Hanfa Zou
Analytica Chimica Acta 2009 Volume 636(Issue 1) pp:34-41
Publication Date(Web):16 March 2009
DOI:10.1016/j.aca.2009.01.033
This study presented an approach to prepare monodisperse immobilized Ti4+ affinity chromatography (Ti4+-IMAC) microspheres for specific enrichment of phosphopeptides in phosphoproteome analysis. Monodisperse polystyrene seed microspheres with a diameter of ca. 4.8 μm were first prepared by a dispersion polymerization method. Monodisperse microspheres with a diameter of ca. 13 μm were prepared using the seed microspheres by a single-step swelling and polymerization method. Ti4+ ion was immobilized after chemical modification of the microspheres with phosphonate groups. The specificity of the Ti4+-IMAC microspheres to phosphopeptides was demonstrated by selective enrichment of phosphopeptides from mixture of tryptic digests of α-casein and bovine serum albumin (BSA) at molar ratio of 1 to 500 by MALDI-TOF MS analysis. The sensitivity of detection for phosphopeptides determined by MALDI-TOF MS was as low as 5 fmol for standard tryptic digest of β-casein. The Ti4+-IMAC microspheres were compared with commercial Fe3+-IMAC adsorbent and homemade Zr4+-IMAC microspheres for enrichment of phosphopeptides. The phosphopeptides and non-phosphopeptides identified by Fe3+-IMAC, Zr4+-IMAC and Ti4+-IMAC methods were 26, 114, 127 and 181, 11, 11 respectively for the same tryptic digest samples. The results indicated that the Ti4+-IMAC had the best performance for enrichment of phosphopeptides.
Co-reporter:Lianghai Hu, Karl-Siegfried Boos, Mingliang Ye, Ren’an Wu, Hanfa Zou
Journal of Chromatography A 2009 Volume 1216(Issue 28) pp:5377-5384
Publication Date(Web):10 July 2009
DOI:10.1016/j.chroma.2009.05.030
As the serum peptidome gets increasing attention for biomarker discovery, one of the important issues is how to efficiently extract the peptides from highly complex human serum for peptidome analysis. Here we developed a fully automated platform for direct injection, on-line extraction, multidimensional separation and MS detection of peptides present in human serum. A capillary SPE column packed with a novel mix mode restricted access material (RAM) exhibiting strong cation exchange and size exclusion chromatography (SCX/SEC) properties were coupled with a nanoliquid chromatography–mass spectrometry (nanoLC-MS) system. The capillary SPE column excludes the high abundant serum proteins such as HSA by size exclusion chromatography and simultaneously extracts the low molecular weight peptides by binding to sulfonic acid residues. Subsequently, the trapped peptides are eluted to a capillary LC column packed with a RP-C18 stationary phase. After injection of only 2 μL human serum to the one-dimensional nanoLC-MS system around 400 peptides could be identified. When conducting a multidimensional separation, the described SCX/SEC/RP-MS platform allows the separation and identification of 1286 peptides present in human serum by the injection and on-line processing of 20 μL human serum sample.
Co-reporter:Yun Wang, Liang Kong, Xiaoyuan Lei, Lianghai Hu, Hanfa Zou, Ed Welbeck, S.W. Annie Bligh, Zhengtao Wang
Journal of Chromatography A 2009 Volume 1216(Issue 11) pp:2185-2191
Publication Date(Web):13 March 2009
DOI:10.1016/j.chroma.2008.05.074
A comprehensive two-dimensional HPLC system with an immobilized liposome chromatography (ILC) column in conjunction with an RP column (in tandem) was developed for the screening and analysis of the membrane-permeable compounds in a traditional Chinese medicine prescription Longdan Xiegan Decoction (LXD). More than 50 components in LXD were resolved using the developed separation system. Eight flavonoids and two iridoids were identified interacting with the ILC column; a system that mimics biomembranes. The results show that the developed comprehensive two-dimensional chromatography system can be used for identifying membrane permeable natural products in complex matrixes such as extracts of traditional Chinese medicine prescriptions.
Co-reporter:Ruijun Tian, Lianbing Ren, Huaijun Ma, Xin Li, Lianghai Hu, Mingliang Ye, Ren’an Wu, Zhijian Tian, Zhen Liu, Hanfa Zou
Journal of Chromatography A 2009 Volume 1216(Issue 8) pp:1270-1278
Publication Date(Web):20 February 2009
DOI:10.1016/j.chroma.2008.10.002
We report the development of a combined strategy for high capacity, comprehensive enrichment of endogenous peptide from complex biological samples at natural pH condition. MCM-41 nanoparticles with highly ordered nanoscale pores (i.e. 4.8 nm) and high-surface area (i.e. 751 m2/g) were synthesized and modified with strong cation-exchange (SCX-MCM-41) and strong anion-exchange (SAX-MCM-41) groups. The modified nanoparticles demonstrated good size-exclusion effect for the adsorption of standard protein lysozyme with molecular weight (MW) of ca. 15 kDa; and the peptides with MW lower than this value can be well adsorbed. Step elution of the enriched peptides with five salt concentrations presented that both modified nanoparticles have high capacity and complementarity for peptides enrichment, and the SAX-MCM-41 nanoparticles has obviously high selectivity for acidic peptides with pI (isoelectric point) lower than 4. Large-scale enrichment of endogenous peptides in 2 mg mouse liver extract was achieved by further combination of SCX-MCM-41 and SAX-MCM-41 with unmodified MCM-41 nanoparticles. On-line 2D nano-LC/MS/MS was applied to analyze the enriched samples, and 2721 unique peptides were identified in total. Two-dimensional analysis of MW versus pI distribution combined with abundance of the identified peptides demonstrated that the three types of nanoparticles have comprehensive complementarity for peptidome enrichment.
Co-reporter:Fangjun Wang, Jing Dong, Mingliang Ye, Ren’an Wu, Hanfa Zou
Journal of Chromatography A 2009 Volume 1216(Issue 18) pp:3887-3894
Publication Date(Web):1 May 2009
DOI:10.1016/j.chroma.2009.02.082
Monolithic columns are widely used in shotgun proteome analysis. However, it is difficult to increase the separation capability and proteome coverage by using conventionally organic polymer-based monolithic column due to the difficulty of controlling homogeneity of the overall pore structure (both pores and microglobules), which leads to relatively low column efficiency. Therefore, we studied the effect of constitute and percentage of porogenic solvent, functional monomer, column length, and separation gradient on the peak capacity and proteome coverage by methacrylate-based reversed phase monolithic columns. It was demonstrated that the porous property of the hydrophobic monolith, which was mainly determined by the porogenic solvent, was crucial to the proteome coverage when similar methacrylate monomer was utilized and a ternary porogenic solvent was adopted to prepare C12 monolithic column with relatively homogeneous overall pore structure. It was also shown that high proteome coverage could be reliably obtained with online multidimensional separation using totally monolithic columns system with the length of analytical column at 85 cm and reversed phase separation gradient at 210 min.
Co-reporter:Xinning Jiang, Xiaoli Dong, Mingliang Ye and Hanfa Zou
Analytical Chemistry 2008 Volume 80(Issue 23) pp:9326
Publication Date(Web):November 4, 2008
DOI:10.1021/ac8017229
The target−decoy database search strategy is often applied to determine the global false-discovery rate (FDR) of peptide identifications in proteome research. However, the confidence of individual peptide identification is typically not determined. In this study, we introduced an approach for the calculation of posterior probability of individual peptide identification from the “local false-discovery rate” (local FDR), which is also determined based on a target−decoy database search. The peptide identification scores output by the database search algorithm were weighted by their discriminating power using a Shannon information entropy based strategy. Then the local FDR of a peptide identification was calculated based on the fraction of decoy identifications among its nearest neighbors within a small space defined by these weighted scores. It was demonstrated that the calculated probability matched the actual probability precisely, and it provided powerful discriminating performance between true positive and false positive identifications. Hence, the sensitivity for peptide identification as well as protein identification was significantly improved when the calculated probability was used to process different proteome data sets. As an instance based strategy, this algorithm provides a safe way for the posterior probability calculation and should work well for data sets with different characteristics.
Co-reporter:Guanghui Han, Mingliang Ye and Hanfa Zou
Analyst 2008 vol. 133(Issue 9) pp:1128-1138
Publication Date(Web):01 Aug 2008
DOI:10.1039/B806775A
Protein phosphorylation is one of the most biologically relevant and ubiquitous post-translational modifications. The analysis of protein phosphorylation is very challenging due to its highly dynamic nature and low stoichiometry. In this article, recent techniques developed for phosphoproteome analysis are reviewed with an emphasis on the new developments in this field in China. To improve the performance of phosphoproteome analysis, many novel methods, either by application of new separation mechanisms or by adoption of new separation materials, were developed to specifically enrich phosphopeptides from complex protein digests. A series of new materials, including nanostructure materials, magnetic materials, and monolithic materials, were applied to prepare immobilized affinity chromatography or metal oxide affinity chromatography to improve the performance of phosphopeptide enrichment. Besides, new software tools were also developed to validate phosphopeptide identification and predict kinase specific phosphorylation sites.
Co-reporter:Fangjun Wang, Jing Dong, Mingliang Ye, Xiaogang Jiang, Ren’an Wu and Hanfa Zou
Journal of Proteome Research 2008 Volume 7(Issue 1) pp:306-310
Publication Date(Web):2017-2-22
DOI:10.1021/pr700562b
A biphasic monolithic capillary column with 10 cm segment of strong-cation exchange monolith and 65 cm segment of reversed-phase monolith was prepared within a single 100 µm i.d. capillary. Separation performance of this column was evaluated by a five-cycle online multidimensional separation of 10 µg tryptic digest of yeast proteins using nanoflow liquid chromatography coupled with tandem mass spectrometry, and it took 12 h for whole separation under the operating pressure only ∼900 psi. Totally, 780 distinct proteins were positively identified through assignment of 2953 unique peptides at false-positive rate less than 1%. The good separation performance of this biphasic column was largely attributed to the good orthogonality of the strong-cation exchange monolith and reversed-phase monolith for multidimensional separation.
Co-reporter:Xinning Jiang, Guanghui Han, Shun Feng, Xiaogang Jiang, Mingliang Ye, Xuebiao Yao and Hanfa Zou
Journal of Proteome Research 2008 Volume 7(Issue 4) pp:1640-1649
Publication Date(Web):2017-2-22
DOI:10.1021/pr700675j
Manual checking is commonly employed to validate the phosphopeptide identifications from database searching of tandem mass spectra. It is very time-consuming and labor intensive as the number of phosphopeptide identifications increases greatly. In this study, a simple automatic validation approach was developed for phosphopeptide identification by combining consecutive stage mass spectrometry data and the target-decoy database searching strategy. Only phosphopeptides identified from both MS2 and its corresponding MS3 were accepted for further filtering, which greatly improved the reliability in phosphopeptide identification. Before database searching, the spectra were validated for charge state and neutral loss peak intensity, and then the invalid MS2/MS3 spectra were removed, which greatly reduced the database searching time. It was found that the sensitivity was significantly improved in MS2/MS3 strategy as the number of identified phosphopeptides was 2.5 times that obtained by the conventional filter-based MS2 approach. Because of the use of the target-decoy database, the false-discovery rate (FDR) of the identified phosphopeptides could be easily determined, and it was demonstrated that the determined FDR can precisely reflect the actual FDR without any manual validation stage.
Co-reporter:Houjiang Zhou, Mingliang Ye, Jing Dong, Guanghui Han, Xinning Jiang, Renan Wu and Hanfa Zou
Journal of Proteome Research 2008 Volume 7(Issue 9) pp:3957-3967
Publication Date(Web):July 17, 2008
DOI:10.1021/pr800223m
The elucidation of protein post-translational modifications, such as phosphorylation, remains a challenging analytical task for proteomic studies. Since many of the proteins targeted for phosphorylation are low in abundance and phosphorylation is typically substoichiometric, a prerequisite for their identification is the specific enrichment of phosphopeptide prior to mass spectrometric analysis. Here, we presented a new method termed as immobilized titanium ion affinity chromatography (Ti4+-IMAC) for enriching phosphopeptides. A phosphate polymer, which was prepared by direct polymerization of monomers containing phosphate groups, was applied to immobilize Ti4+ through the chelating interaction between phosphate groups on the polymer and Ti4+. The resulting Ti4+-IMAC resin specifically isolates phosphopeptides from a digest mixture of standard phosphoproteins and nonphosphoprotein (BSA) in a ratio as low as 1:500. Ti4+-IMAC was further applied for phosphoproteome analysis of mouse liver. We also compared Ti4+-IMAC to other enrichment methods including Fe3+-IMAC, Zr4+-IMAC, TiO2 and ZrO2, and demonstrate superior selectivity and efficiency of Ti4+-IMAC for the isolation and enrichment of phosphopeptides. The high specificity and efficiency of phosphopeptide enrichment by Ti4+-IMAC mainly resulted from the flexibility of immobilized titanium ion with spacer arm linked to polymer beads as well as the specific interaction between immobilized titanium ion and phosphate group on phosphopeptides.
Co-reporter:Fangjun Wang;Mingliang Ye;Jing Dong;Ruijun Tian;Lianghai Hu;Guanghui Han;Xinning Jiang;Ren'an Wu
Journal of Separation Science 2008 Volume 31( Issue 14) pp:2589-2597
Publication Date(Web):
DOI:10.1002/jssc.200800181
Abstract
The postcolumn void volume, which is introduced by the connecting tubing and void ESI emitter in the nanoflow LC coupled with MS/MS system (μLC-MS/MS), is harmful for the analysis of peptides in shotgun proteome analysis. A new type of porous C12 monolithic ESI emitter was prepared to eliminate the disruption and mixing effects occurring in the connecting tubing and void emitter. It was demonstrated that the porous hydrophobic monolith inside the emitter played a key role in retaining the good peak profile, and the average peak capacity of the whole separation system increased 12.8% in contrast to commercially available void emitter. Then, the porous C12 monolithic emitter was applied in label-free quantitative proteome analysis of two standard protein mixtures that were spiked into the tryptic digest of mouse livers extract. Compared to commercially available void ESI emitter, the number of proteins with reliable results in quantification increased greatly. And the relative quantities of the four standard proteins were all determined with the relative error ⪇6.8%. However, quantitative information of only three standard proteins could be obtained when void emitter was used.
Co-reporter:Xiao-yuan LEI, Liang KONG, Xing-ye SU, Ming GUO, Han-fa ZOU
Chemical Research in Chinese Universities 2008 Volume 24(Issue 4) pp:411-419
Publication Date(Web):July 2008
DOI:10.1016/S1005-9040(08)60087-2
Abstract
Some natural products, such as traditional Chinese medicines(TCMs), contain compounds with anticanccr activity and have attracted a great interest in recent years as alternative anticanccr therapies. A quick and convenient assay for screening antimicrotubule compounds in which in vitro microdialysis/high-performance liquid chromatography (HPLC) is used to monitor the binding of the compounds extracted from TCM Taxus cuspidata Siebold & Zucc(Taxus) to microtubuics is reported. It was observed that the extract of Taxus contains at least five compounds which have affinity interaction with microtubuics by biological fingerprinting analysis, and they were identified as the taxoids of taxol, baccatin III, 10-deacetylbaccatin III(10-DAB), cephalomannine and 7-epi-10-deacetyltaxol (7-epi-10-DAT) based on the comparison of their high-performance liquid chromatographic/mass spectrometric and UV spectra with those of the standard samples, both assembly-promoting and disassembly-inhibiting characteristics of those compounds were evaluated. It was observed that baccatin III and 10-DAB bound to microtubules and the binding degrees were influenced by GTP. Competitive binding behavior of taxol with other four taxoids to microlubulcs was also investigated.
Co-reporter:Liang Zhao;Ren’an Wu;Guanghui Han
Journal of The American Society for Mass Spectrometry 2008 Volume 19( Issue 8) pp:1176-1186
Publication Date(Web):2008 August
DOI:10.1016/j.jasms.2008.04.027
The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe3O4/SiO2 core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol β-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe3O4 nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe3O4 nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of α-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.
Co-reporter:Xiaoli Dong, Ren’an Wu, Jing Dong, Minghuo Wu, Yan Zhu, Hanfa Zou
Journal of Chromatography B 2008 Volume 875(Issue 1) pp:317-322
Publication Date(Web):1 November 2008
DOI:10.1016/j.jchromb.2008.05.019
A chiral capillary monolithic column for capillary electrochromatography (CEC) was prepared by covalent bonding of cellulose tris(3,5-dimethylphenylcarbamate) (CDMPC) on the silica monolithic matrix within the confine of a 50-μm i.d. bare fused silica capillary. Several pairs of enantiomers including neutral and basic analytes were baseline resolved on the newly prepared chiral capillary monolithic column in CEC with aqueous mobile phases. Fast enantioseparation was achieved due to the favorable dynamic properties of silica monolith. The covalent bonding of CDMPC as the chiral stationary phase for CEC also enabled the use of THF in mobile phase for enantioseparation of prazquantel by overcoming the incompatibility of THF and the physically coated CDMPC on a column.
Co-reporter:Lianghai Hu, Mingliang Ye, Xiaogang Jiang, Shun Feng, Hanfa Zou
Analytica Chimica Acta 2007 Volume 598(Issue 2) pp:193-204
Publication Date(Web):29 August 2007
DOI:10.1016/j.aca.2007.07.046
Proteomics is defined as the analysis of part or all of the protein components of a complex biological system (a cell, organ or tissue) at a given moment. Due to the huge number of proteins encoded by the genome, novel analytical techniques must be developed to meet the need of large scale analysis. This has led to the hyphenation of multiple techniques to achieve this object. Here current status of the hyphenated analytical techniques of one-dimensional and multidimensional liquid chromatography–mass spectrometry for shotgun proteomic analysis is reviewed, and on-line techniques for automated sample preparation and injection are also covered. In addition, the hyphenated techniques for peptidome analysis are also covered.
Co-reporter:Songyun Xu;Chensong Pan;Xin Li;Houjiang Zhou;Xiaogang Jiang;Mingliang Ye;Yu Zhang
Journal of Separation Science 2007 Volume 30(Issue 6) pp:930-943
Publication Date(Web):20 MAR 2007
DOI:10.1002/jssc.200600479
Human plasma contains a complex matrix of proteolytically derived peptides (plasma peptidome) that may provide a correlate of biological events occurring in the entire organism. Analyzing these peptides from a small amount of serum/plasma is difficult due to the complexity of the sample and the low levels of these peptides. Here, we describe a novel peptidome analysis approach using multiwalled carbon nanotubes (MWCNTs) as an alternative adsorbent to capture endogenous peptides from human plasma. Harvested peptides were analyzed by using liquid chromatography-mass spectrometry as a means of detecting and assessing the adsorbed molecules. The improved sensitivity and resolution obtained by using liquid chromatography-mass spectrometry allowed detection of 2521 peptide features (m/z 300–1800 range) in about 50 μL of plasma. 374 unique peptides were identified with high confidence by two-dimensional liquid chromatography system coupled to a nano-spray ionization linear ion trap-mass spectrometer. High recovery of BSA digest peptides enriched with MWCNTs, in both standard buffer and high abundance protein solution, was observed. Comparative studies showed that MWCNTs were superior to C18 and C8 for the capture of the smaller peptides. This approach could hold promise of routine plasma peptidome analysis.
Co-reporter:Chuanhui Xie;Jiwei Hu;Ruijun Tian;Zhike He
Journal of Separation Science 2007 Volume 30(Issue 6) pp:891-899
Publication Date(Web):27 MAR 2007
DOI:10.1002/jssc.200600173
A novel monolithic silica column with zwitterionic stationary phase was prepared by in-situ covalent attachment of phenylalanine to a 3-glycidoxypropyltriethoxysilane-modified silica monolith. Due to the zwitterionic nature of the resulting stationary phase, the density and sign of the net surface charge, and accordingly the direction and magnitude of electroosmotic flow in this column during capillary electrochromatography could be manipulated by adjusting the pH values of the mobile phase. CEC separations of various acidic and basic compounds were performed on the prepared column in anodic and weakly cathodic EOF modes, respectively. The peak tailing of basic compounds in CEC on a silica column could be alleviated at optimized buffer compositions. Besides the electrophoretic mechanism and weak hydrophobic interaction, weak cation- and anion-exchange interactions are also involved in the separations of acids and bases, respectively, on the zwitterionic column.
Co-reporter:Ruijun Tian;Mingliang Ye;Lianghai Hu;Xin Li
Journal of Separation Science 2007 Volume 30(Issue 14) pp:2204-2209
Publication Date(Web):7 AUG 2007
DOI:10.1002/jssc.200700156
In this study, an improved method for human plasma peptidome analysis including selective porous silica nanoparticles (MCM-41) extraction and subsequent online 2-D nano-LC-MS/MS analysis was established. Enhanced enrichment efficiency for the MCM-41 extraction was obtained by adjusting the pH of the plasma sample to 2.5. A total of 1680 unique peptides were identified in the plasma sample obtained from one healthy donor, which is nearly twice the amount identified from the native state of the plasma sample. The hydrophobic property, molecular weight (MW), and pI distribution of the identified peptides at pH 2.5 and native state of the plasma sample were systematically investigated and compared. Furthermore, many unusual cleaved peptides from plasma proteins (e. g., HSA) were observed at pH 2.5, which clearly show a ladder pattern. The cleavage patterns for all of the identified peptides at pH 2.5 were summarized, and chymosin and cathepsin D were confirmed as the possible peptidases responsible for the change of cleavage pattern in peptide profiling.
Co-reporter:Houjiang Zhou;Jing Dong;Renan Wu;Mingliang Ye
Journal of Separation Science 2007 Volume 30(Issue 17) pp:2917-2923
Publication Date(Web):8 OCT 2007
DOI:10.1002/jssc.200700350
A method to prepare zirconium phosphate (ZrP)-modified monolithic capillary column for highly specific capture of phosphopeptides is presented. In this method, the phosphate monolithic capillary column was prepared by direct copolymerization of the functional monomer containing phosphate group (ethylene glycol methacrylate phosphate) and cross-linker (bis-acrylamide) in a ternary porogenic solvent. Copolymerization of cross-linker and functional monomer simplifies the procedure for the preparation of phosphate chromatographic media. After Zr4+ was immobilized, the ZrP-modified monolithic capillary column was evaluated by the analysis of standard phosphoproteins and the excellent selectivity of this approach was demonstrated by analyzing phosphopeptides in the digest mixture of β-casein and BSA with molar ratio of 1:200.
Co-reporter:Mingliang Ye;Jing Dong;Xiaoli Dong;Renan Wu;Junjie Ou
Journal of Separation Science 2007 Volume 30(Issue 17) pp:2986-2992
Publication Date(Web):10 OCT 2007
DOI:10.1002/jssc.200700402
A CEC monolithic column with strong cation-exchange (SCX) stationary phase based on hydrophilic monomers was prepared by in situ polymerization of acrylamide, methylenebisacrylamide, and 2-acrylamido-2-methyl-1-propanesulfonic acid (AMPS) in a complete organic binary porogenic solvent consisting of DMSO and dodecanol. The sulfonic groups provided by the monomer AMPS on the surface of the stationary phase generate an EOF from anode to cathode, and serve as an SCX stationary phase at the same time. The monolithic stationary phase exhibited normal-phase chromatographic behavior for neutral analytes. For charged analytes, electrostatic interaction/repulsion with the monolith was observed. The strong SCX monolithic column has been successfully employed in the electrochromatographic separation of basic drugs, peptides, and alkaloids extracted from natural products.
Co-reporter:Ruijun Tian;He Zhang;Mingliang Ye Dr.;Xiaogang Jiang;Lianghai Hu;Xin Li;Xinhe Bao Dr. Dr.
Angewandte Chemie 2007 Volume 119(Issue 6) pp:
Publication Date(Web):13 DEC 2006
DOI:10.1002/ange.200603917
Poren zeigen Wirkung: Mit Siliciumoxidpartikeln mit einer Porengröße von 20.5 Å (siehe TEM-Bild) gelingt die Anreicherung von Peptiden mit Molekulargewichten von 1 bis 12 kDa in Humanplasma; andere Plasmaproteine werden dagegen nicht angereichert. Durch seine Porenstruktur ist das Material besser für die Peptidanreicherung geeignet als Adsorbens- und Ultrafiltrationsmethoden.
Co-reporter:Ruijun Tian;He Zhang;Mingliang Ye Dr.;Xiaogang Jiang;Lianghai Hu;Xin Li;Xinhe Bao Dr. Dr.
Angewandte Chemie International Edition 2007 Volume 46(Issue 6) pp:
Publication Date(Web):13 DEC 2006
DOI:10.1002/anie.200603917
Pores for effect: Silica particles with a pore size of 20.5 Å (see TEM image) are effective for enriching peptides in human plasma over a wide range of molecular weights from 1 to 12 kDa, while repelling most other plasma proteins outside. The pore structure of the material makes it superior for peptide enrichment compared to adsorbent- and ultrafiltration-based methods.
Co-reporter:Hua Xiao, Xin Li, Hanfa Zou, Ling Yang, Yanqin Yang, Yulin Wang, Hailin Wang, X. Chris Le
Analytica Chimica Acta 2006 Volume 556(Issue 2) pp:340-346
Publication Date(Web):25 January 2006
DOI:10.1016/j.aca.2005.09.051
The cellular mechanism based on P-glycoprotein (PGP) for its drug pump function has become very important in multidrug resistance (MDR) research. A method has been established to characterize PGP on single K562 cell by coupling capillary electrophoresis with laser induced fluorescence detection. A permeable intact cell after the immunoassay binding with fluorescence labeling antibody was injected into the capillary and directly separated without lysis. It was found that once 5–10 optional cells were detected in batch, the PGP amount on this cell line could be outlined and calculated clearly. The PGP amount on K562 MDR cell line is 3.88 times higher than that on K562 sensitive cell line. These two cell lines with immunoassay binding were also analyzed by injection of multi-cells in order to improve the throughput. A resistance factor so called multidrug resistance multiple (MRM) was introduced to evaluate the MDR difference between cell lines. The MRM values of the cell line K562 measured by single cell analysis are well correlated with those by flow cytometry, which also prove the validity of our method in single cell analysis for the possibility of cancer diagnosis, pharmacokinetics and drug screening in future.
Co-reporter:Lianghai Hu;Shun Feng;Xin Li;Liang Kong;Xingye Su;Xueguo Chen;Mingliang Ye;Feng Qin
Journal of Separation Science 2006 Volume 29(Issue 6) pp:881-888
Publication Date(Web):13 MAR 2006
DOI:10.1002/jssc.200500442
A mode of comprehensive 2-D LC was developed by coupling a silica-bonded HSA column to a silica monolithic ODS column. This system combined the affinity property of the HSA column and the high-speed separation ability of the monolithic ODS column. The affinity chromatography with HSA-immobilized stationary phase was applied to study the interaction of multiple components in traditional Chinese medicines (TCMs) with HSA according to their affinity to protein in the first dimension. Then the unresolved components retained on the HSA column were further separated on the silica monolithic ODS column in the second dimension. By hyphenating the 2-D separation system to diode array detector and MS detectors, the UV and molecular weight information of the separated compounds can also be obtained. The developed separation system was applied to analysis of the extract of Rheum palmatum L., a number of low-abundant components can be separated on a single peak from the HSA column after normalization of peak heights. Six compounds were preliminarily identified according to their UV and MS spectra. It showed that this system was very useful for biological fingerprinting analysis of the components in TCMs and natural products.
Co-reporter:Chuanhui Xie;Feng Qin;Zhiyuan Yu;Liang Kong;Mingliang Ye
Journal of Separation Science 2006 Volume 29(Issue 10) pp:1332-1343
Publication Date(Web):8 JUN 2006
DOI:10.1002/jssc.200600030
Monolithic materials have become a well-established format for stationary phases in the field of capillary electrochromatography. Four types of monoliths, namely particle-fixed, silica-based, polymer-based, and molecularly imprinted monoliths, have been utilized as enantiomer-selective stationary phases in CEC. This review summarizes recent developments in the area of monolithic enantiomer-selective stationary phases for CEC. The preparative procedure and the characterization of these columns are highlighted. In addition, the disadvantages and limitations of different monolithic enantiomer-selective stationary phases in CEC are briefly discussed.
Co-reporter:Xueguo Chen, Lianghai Hu, Xingye Su, Liang Kong, Mingliang Ye, Hanfa Zou
Journal of Pharmaceutical and Biomedical Analysis 2006 Volume 40(Issue 3) pp:559-570
Publication Date(Web):24 February 2006
DOI:10.1016/j.jpba.2005.07.043
A hyphenated method for the isolation and identification of components in a traditional Chinese medicine of Honeysuckle was developed. Ion-exchange chromatography (IEC) was chosen for the fractionation of Honeysuckle extract, and then followed by concentration of all the fractions with rotary vacuum evaporator. Each of the enriched fractions was then further analyzed by reversed-phase liquid chromatography-atmospheric pressure chemical ionization mass spectrometer (RPLC-APCI/MS) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) with matrix of oxidized carbon nanotubes, respectively. It can be noted totally more than 117 components were detected by UV detector, APCI/MS and MALDI-TOF/MS in Honeysuckle extract except the 145 components identified by MALDI-TOF/MS alone with this integrated approach, and 7 of them were preliminary identified according to their UV spectra and mass spectra performed by APCI/MS and MALDI-TOF/MS, respectively. The obtained analytical results not only indicated the approach of integration IEC fractionation with RPLC-APCI/MS and MALDI-TOF/MS is capable of analyzing complex samples, but also exhibited the potential power of the mass spectrometer in detection of low-mass compounds, such as traditional Chinese medicines (TCMs) and complex biological samples.
Co-reporter:Liang Kong;Shouwan Tang;Yueqi Liu;Junjie Ou;Xin Li
Journal of Molecular Recognition 2006 Volume 19(Issue 1) pp:39-48
Publication Date(Web):2 NOV 2005
DOI:10.1002/jmr.756
β-Cyclodextrin (β-CyD) was cross-linked by hexamethylene diisocyanate and the polymer was investigated for adsorption of aromatic amino acids (AAA) from phosphate buffer. High adsorption rates were observed at the beginning and the adsorption equilibrium was then gradually achieved in about 45 min. The adsorption of AAA decreased with the increase of initial concentration and also temperature. Under the same conditions, the adsorption efficiencies of AAA were in the order of L-tryptophan (L-Trp) > L-phenylalanine (L-Phe) > L-tyrosine (L-Tyr). Much higher adsorption values, up to 52.4 and 43.0 mg/g for L-Trp and L-Phe, respectively, at 50 mmol/L and 3.2 mg/g for L-Tyr at 2 mmol/L, were obtained with the β-CyD polymer at 37 °C. It was shown that the adsorption of AAA on the β-CyD polymer was consistent with the Freundlich isotherm equation. The adsorption of mixed aromatic amino acids and branched-chain amino acids (BCAA) showed that AAA were preferentially adsorbed with adsorption efficiencies 10–24%, while those of BCAA were lower than 2%. It seems that the structure and hydrophobicity of amino acid molecules are responsible for the difference in adsorption, by influencing the strength of interactions between amino acid molecule and the polymer. Copyright © 2005 John Wiley & Sons, Ltd.
Co-reporter:Junjie Ou, Lianghai Hu, Ligang Hu, Xin Li, Hanfa Zou
Talanta 2006 Volume 69(Issue 4) pp:1001-1006
Publication Date(Web):15 June 2006
DOI:10.1016/j.talanta.2005.12.003
The bisphenol A (BPA) imprinted monolithic precolumn has been prepared by in situ polymerization using 4-vinylpyridine (4-VP) and ethylene dimethacrylate (EDMA) as functional monomer and cross-linker, respectively. The column with good flow-through property was obtained by changing the molar ratio of the porogens (toluene and dodecanol). The selectivity and retention properties of the monolith for the BPA and other phenolic compounds were evaluated. The results show that the hydrophobic and hydrogen-bonding interaction plays important roles in the recognition process. The determination of BPA and other phenolic compounds with on-line solid-phase extraction (SPE) by monolithic precolumn coupled with conventional particulates packed and monolithic reversed-phase columns, respectively, was performed. The method was successfully applied to the analysis of phenolic compounds in river water.
Co-reporter:Xingye Su, Liang Kong, Xin Li, Xueguo Chen, Ming Guo, Hanfa Zou
Journal of Chromatography A 2005 Volume 1076(1–2) pp:118-126
Publication Date(Web):27 May 2005
DOI:10.1016/j.chroma.2005.04.031
Biofingerprinting chromatogram analysis, which is defined as the comparison of fingerprinting chromatograms of the extract of traditional Chinese medicines (TCMs) before and after the interaction with biological systems (DNA, protein, cell, etc.), was proposed for screening and analysis of the multiple bioactive compounds in TCMs. A method of microdialysis sampling combined with high performance liquid chromatography (HPLC) was applied to the study of DNA-binding property for the extracts of TCMs. Seven compounds were found to bind to calf thymus DNA (ct-DNA) from the TCMs of Coptis chinensis Franch (Coptis), but only three ones from Phellodendron amurense Rupr. (Phellodendron) and none from Sophora flavescens Ait. (Sophora) to bind to ct-DNA, respectively. Three of them were identified as berberine, palmatine and jatrorrhizine and their association constants (K) to ct-DNA were determined by microdialysis/HPLC. Competitive binding behaviors of them to ct-DNA were also investigated.
Co-reporter:Chensong Pan, Songyun Xu, Hanfa Zou, Zhong Guo, Yu Zhang, Baochuan Guo
Journal of the American Society for Mass Spectrometry 2005 Volume 16(Issue 2) pp:263-270
Publication Date(Web):February 2005
DOI:10.1016/j.jasms.2004.11.005
A method with carbon nanotubes functioning both as the adsorbent of solid-phase extraction (SPE) and the matrix for matrix assisted laser desorption/ionization mass spectrometry (MALDI-MS) to analyze small molecules in solution has been developed. In this method, 10 μL suspensions of carbon nanotubes in 50% (vol/vol) methanol were added to the sample solution to extract analytes onto surface of carbon nanotubes because of their dramatic hydrophobicity. Carbon nanotubes in solution are deposited onto the bottom of tube with centrifugation. After removing the supernatant fluid, carbon nanotubes are suspended again with dispersant and pipetted directly onto the sample target of the MALDI-MS to perform a mass spectrometric analysis. It was demonstrated by analysis of a variety of small molecules that the resolution of peaks and the efficiency of desorption/ionization on the carbon nanotubes are better than those on the activated carbon. It is found that with the addition of glycerol and sucrose to the dispersant, the intensity, the ratio of signal to noise (S/N), and the resolution of peaks for analytes by mass spectrometry increased greatly. Compared with the previously reported method by depositing sample solution onto thin layer of carbon nanotubes, it is observed that the detection limit for analytes can be enhanced about 10 to 100 times due to solid-phase extraction of analytes in solution by carbon nanotubes. An acceptable result of simultaneously quantitative analysis of three analytes in solution has been achieved. The application in determining drugs spiked into urine has also been realized.
Co-reporter:Jing Dong;Chuanhui Xie;Xingye Su;Jiwei Hu;Hua Xiao;Ruijun Tian;Zhike He
Journal of Separation Science 2005 Volume 28(Issue 8) pp:751-756
Publication Date(Web):1 APR 2005
DOI:10.1002/jssc.200400101
A monolithic silica based strong cation-exchange stationary phase was successfully prepared for capillary electrochromatography. The monolithic silica matrix from a sol-gel process was chemically modified by treatment with 3-mercaptopropyltrimethoxysilane followed by a chemical oxidation procedure to produce the desired function. The strong cation-exchange stationary phase was characterized by its substantial and stable electroosmotic flow (EOF), and it was observed that the EOF value of the prepared column remained almost unchanged at different buffer pH values and slowly decreased with increasing phosphate concentration in the mobile phase. The monolithic silica column with strong cation-exchange stationary phase has been successfully employed in the electrochromatographic separation of β-blockers and alkaloids extracted from traditional Chinese medicines (TCMs). The column efficiencies for the tested β-blockers varied from 210,000 to 340,000 plates/m. A peak compression effect was observed for atenolol with the mobile phase having a low phosphate concentration.
Co-reporter:Junjie Ou;Shouwan Tang
Journal of Separation Science 2005 Volume 28(Issue 17) pp:2282-2287
Publication Date(Web):26 OCT 2005
DOI:10.1002/jssc.200500165
Two molecular imprinting polymer (MIP) monolithic columns with (S)-(–)-1,1′-bi-2-naphthol and (R)-(+)-5,5′,6,6′,7,7′,8,8′-octahydro-1,1′-bi-2-naphthol as the templating molecules, respectively, have been prepared by in situ polymerization using 4-vinylpyridine and ethylene dimethacrylate as functional monomer and cross-linker, respectively. The columns with good flow-through properties were obtained by changing the molar ratio of the functional monomer and the template molecule. The effects of mobile-phase composition on separation of enantiomers were systematically investigated. The results indicate that hydrophobic interaction in aqueous solution and hydrogen-bonding interaction in ACN between the enantiomers and polymers could play important roles in the retention and resolution. The effects of chromatographic conditions, such as flow rate, column temperature, sample loading, on the enantioseparation were also studied. Further, these two MIP columns show a cross-reactivity.
Co-reporter:Qin Feng;Chen Xiao-Ming;Liu Yue-Qi;Zou Han-Fa;Wang Jun-De
Chinese Journal of Chemistry 2005 Volume 23(Issue 7) pp:
Publication Date(Web):16 AUG 2005
DOI:10.1002/cjoc.200590885
The classical method for preparation of covalently boned cellulose derivative chiral stationary phases (CSP) with diisocyanate as spacer was improved. Diisocyanate was firstly allowed to react with 3-aminopropyltriethoxysilane, and the resulting product was then applied as the spacer reagent to immobilize cellulose derivatives onto silica gel. Influences of the amount and the length of the spacer on the optical resolution ability of the CSP were investigated. Comparing improved procedure to classical diisocyanate method, the cross-linking between the glucose units of the cellulose derivatives was avoided to the most extent. With the improved procedure, regio-nonselective ways could be adopted to prepare covalently bonded CSP, which showed an advantage for the rapid preparation.
Co-reporter:Xiaoming Chen;Yueqi Liu;Liang Kong;Feng Qin
Journal of Separation Science 2004 Volume 27(Issue 14) pp:1195-1201
Publication Date(Web):30 SEP 2004
DOI:10.1002/jssc.200401789
Dimethyl dicarboxy α-biphenyl (DDB) and its analogues represent atropisomers which have been resolved on the covalently bonded cellulose tris-(3,5-dimethylphenylcarbamate) (CDMPC) CSP. Different kinds of alcohols, tetrahydrofuran (THF), and chloroform were employed as mobile phase modifiers (MPMs), and their influence on the retention and separation of the enantiomers was investigated. Ternary mobile phases (hexane/2-propanol/THF, hexane/2-propanol/chloroform) were employed to investigate the separation of the five enantiomers. The advantages of the broader choice of solvents offered by the covalently bonded CDMPC CSP were discussed. The effect of structural variation of the enantiomers on their retention and separation was investigated.
Co-reporter:Liang Zhao, Ren'an Wu, Guanghui Han, Houjiang Zhou, Lianbing Ren, Ruijun Tian, Hanfa Zou
Journal of the American Society for Mass Spectrometry (August 2008) Volume 19(Issue 8) pp:1176-1186
Publication Date(Web):1 August 2008
DOI:10.1016/j.jasms.2008.04.027
The highly selective capture of phosphopeptides from proteolytic digests is a great challenge for the identification of phosphoproteins by mass spectrometry. In this work, the zirconium phosphonate-modified magnetic Fe3O4/SiO2 core/shell nanoparticles have been synthesized and successfully applied for the selective capture of phosphopeptides from complex tryptic digests of proteins before the analysis of MALDI-TOF mass spectrometry with the desired convenience of sample handling. The ratio of magnetic nanoparticle to protein and the incubation time for capturing phosphopeptides from complex proteolytic digests were investigated, and the optimized nanoparticle-to-protein ratio and incubation time were between 15:1 to 30:1 and 30 min, respectively. The excellent detection limit of 0.5 fmol β-casein has been achieved by MALDI-TOF mass spectrometry with the specific capture of zirconium phosphonate-modified magnetic Fe3O4 nanoparticles. The great specificity of zirconium phosphonate-modified magnetic Fe3O4 nanoparticles to phosphopeptides was demonstrated by the selective capture of phosphopeptides from a complex tryptic digest of the mixture of α-casein and bovine serum albumin at molar ratio of 1 to 100 in MALDI-TOF-MS analysis. An application of the magnetic nanoparticles to selective capture phosphopeptides from a tryptic digest of mouse liver lysate was further carried out by combining with nano-LC-MS/MS and MS/MS/MS analyses, and a total of 194 unique phosphopeptides were successfully identified.
Co-reporter:Junjie Ou, Jing Dong, Tuijun Tian, Jiwei Hu, ... Hanfa Zou
Journal of Biochemical and Biophysical Methods (23 February 2007) Volume 70(Issue 1) pp:71-76
Publication Date(Web):23 February 2007
DOI:10.1016/j.jbbm.2006.07.003
Two chiral compounds, Tröger's base and tetrahydropalmatine, were enantioseparated on the (5S, 11S)-(-)-Tröger's base and l-tetrahydropalmatine imprinted monolithic capillary columns with CEC, respectively. The monoliths were prepared by in situ thermal-initiated copolymerization of methacrylic acid (MAA) and ethylene dimethacrylate (EDMA). After optimizing the ratio of porogens (toluene and dodecanol), the obtained monolithic capillary columns show good flow-through property and enantioselectivity. The influences of CEC parameters such as pH of the buffer, organic solvent and salt concentration on the electroosmotic flow (EOF) and recognition selectivity were systematically investigated. Under the optimal conditions, baseline resolutions of two chiral compounds were achieved. In addition, the fast separation was obtained within 4 min by applying higher voltage and assisting pressure of 6 bar.
Co-reporter:Yue Wu, Fangjun Wang, Zheyi Liu, Hongqiang Qin, Chunxia Song, Junfeng Huang, Yangyang Bian, Xiaoluan Wei, Jing Dong and Hanfa Zou
Chemical Communications 2014 - vol. 50(Issue 14) pp:NaN1710-1710
Publication Date(Web):2013/12/09
DOI:10.1039/C3CC47998F
Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.
Co-reporter:Zhongshan Liu, Junjie Ou, Hui Lin, Zheyi Liu, Hongwei Wang, Jing Dong and Hanfa Zou
Chemical Communications 2014 - vol. 50(Issue 66) pp:NaN9290-9290
Publication Date(Web):2014/06/12
DOI:10.1039/C4CC03451A
Hybrid monoliths with a macroporous structure were prepared within a few minutes via a photoinduced thiol–ene polymerization reaction, the surfaces of which showed hydrophobic character. The monolithic column demonstrated good separation performance towards alkylbenzenes, peptides, proteins and BSA tryptic digest in cLC.
Co-reporter:Hao Wan, Junfeng Huang, Zhongshan Liu, Jinan Li, Weibing Zhang and Hanfa Zou
Chemical Communications 2015 - vol. 51(Issue 45) pp:NaN9394-9394
Publication Date(Web):2015/04/30
DOI:10.1039/C5CC01980J
Combining high surface area, numerous active sites, strong magnetic response, multivalent synergistic binding effects and outstanding hydrophilicity, a novel hydrophilic composite demonstrated efficient and selective enrichment of glycopeptides from the complex sample.
Co-reporter:Jin Chen, Zheyi Liu, Fangjun Wang, Jiawei Mao, Ye Zhou, Jing Liu, Hanfa Zou and Yukui Zhang
Chemical Communications 2015 - vol. 51(Issue 79) pp:NaN14760-14760
Publication Date(Web):2015/08/13
DOI:10.1039/C5CC06072A
We develop an acidic vapor assisted electrospray ionization strategy within an enclosed electrospray ionization source to counteract the ion suppression effects caused by trifluoroacetic acid. The mass spectrometry signal intensity of intact proteins was improved 10 times and the number of valid signals for E. coli intact protein samples was improved 96% by using this strategy.
Co-reporter:Zhenzhen Deng, Mingliang Ye, Yangyang Bian, Zheyi Liu, Fangjie Liu, Chunli Wang, Hongqiang Qin and Hanfa Zou
Chemical Communications 2014 - vol. 50(Issue 90) pp:NaN13962-13962
Publication Date(Web):2014/09/11
DOI:10.1039/C4CC04906C
A simple, cost-effective and high throughput method was developed for multiplexed kinase activity assay based on the multiplex isotope labeling of designed substrate peptides. This strategy was successfully applied to monitor the time-dependent consumption of substrates and generation of products in the single and multiple substrate systems.
Co-reporter:Hongqiang Qin, Fangjun Wang, Peiyuan Wang, Liang Zhao, Jun Zhu, Qihua Yang, Ren'an Wu, Mingliang Ye and Hanfa Zou
Chemical Communications 2012 - vol. 48(Issue 7) pp:NaN963-963
Publication Date(Web):2011/11/03
DOI:10.1039/C1CC15222J
Ti4+-EPO nanoparticles were adopted as the adsorbent for in situ solid phase enrichment and isotope labeling of endogenous phosphopeptides, which has great potential application in high-throughput analyses of biological samples for screening and discovery of disease-specific biomarkers.
Co-reporter:Bo Xu, Lipeng Zhou, Fangjun Wang, Hongqiang Qin, Jun Zhu and Hanfa Zou
Chemical Communications 2012 - vol. 48(Issue 12) pp:NaN1804-1804
Publication Date(Web):2011/12/06
DOI:10.1039/C2CC16662C
Hierarchical Ti-aluminophosphate-5 molecular sieves templated by glucose have been synthesized and applied as a potential adsorbent for the first time to selectively capture phosphopeptides from complex peptide mixtures prior to MALDI-TOF MS analysis.
Co-reporter:Zhichao Xiong, Liang Zhao, Fangjun Wang, Jun Zhu, Hongqiang Qin, Ren'an Wu, Weibing Zhang and Hanfa Zou
Chemical Communications 2012 - vol. 48(Issue 65) pp:NaN8140-8140
Publication Date(Web):2012/06/25
DOI:10.1039/C2CC33600F
Hybrid Fe3O4@SiO2@PEG-Maltose MNPs were synthesized by SI-ATRP of branched PEG brushes on the surface and subsequent functionalization with hydrophilic maltose group, and the multifunctional materials were utilized for selective enrichment of N-linked glycopeptides from biological samples with high specifity, high sensitivity, and large binding capacity.
Co-reporter:Liang Zhao, Hongqiang Qin, Zhengyan Hu, Yi Zhang, Ren'an Wu and Hanfa Zou
Chemical Science (2010-Present) 2012 - vol. 3(Issue 9) pp:NaN2838-2838
Publication Date(Web):2012/06/19
DOI:10.1039/C2SC20363D
Immobilized metal affinity chromatography (IMAC) is a powerful method in phosphopeptide enrichment. However, the achievement of highly specific enrichment and sensitive detection of phosphopeptide by IMAC is still a big challenge because of the lack of high specificity and large binding capacity of conventional IMAC materials. Here, we report a novel IMAC nanoparticle to dramatically improve the enrichment specificity for the phosphopeptide by introducing a titanium phosphate moiety on a poly(ethylene glycol) methacrylate (PEG) brush decorated Fe3O4@SiO2 core–shell nanoparticle (denoted as Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle). The thicker grafting layer of the PEG brushes has a higher chelating capacity of titanium ions. Due to the combination of the superior nonfouling property and the enhanced binding capacity of the grafted PEG brushes, the Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle demonstrated a high phosphopeptide recovery (over 70%) and low limit of detection (0.5 fmol), along with an exceptional great specificity to capture phosphopeptides from a tryptic digest of the mixture of a nonphosphorylated protein BSA and a phosphorylated protein α-casein with molar ratios of BSA/α-casein up to 2000:1. In the analysis of a real complex biological sample, the tryptic digests of Arabidopsis, 2447 unique phosphopeptides have been identified, showing a superior performance of the Fe3O4@SiO2@PEG–Ti4+ IMAC nanoparticle than that of Fe3O4@SiO2–Ti4+ (1186) and commercial TiO2 microspheres (961). We believe that the PEG decoration for IMAC materials will be a convenient approach to significantly improve the specificity and the binding capacity of phosphopeptide enrichment.
Co-reporter:Zhichao Xiong, Lingyi Zhang, Chunli Fang, Quanqing Zhang, Yongsheng Ji, Zhang Zhang, Weibing Zhang and Hanfa Zou
Journal of Materials Chemistry A 2014 - vol. 2(Issue 28) pp:NaN4480-4480
Publication Date(Web):2014/05/08
DOI:10.1039/C4TB00479E
Highly selective and efficient enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel immobilized metal affinity chromatography (IMAC) material has been synthesized to improve the enrichment specificity and sensitivity for phosphopeptides by introducing a titanium phosphate moiety on a multilayer polysaccharide (hyaluronate (HA) and chitosan (CS)) coated Fe3O4@SiO2 nanoparticle (denoted as Fe3O4@SiO2@(HA/CS)10–Ti4+ IMAC). The thicker multilayer polysaccharide endows excellent hydrophilic properties and a higher binding capacity of the titanium ion to the IMAC material. Due to the combination of uniform magnetic properties, highly hydrophilic properties and enhanced binding capacity of the titanium ion, the Fe3O4@SiO2@(HA/CS)10–Ti4+ nanoparticle possesses many merits, such as high selectivity for phosphopeptides (phosphopeptides/non-phosphopeptides at a molar ratio of 1:2000), extreme detection sensitivity (0.5 fmol), large binding capacity (100 mg g−1), high enrichment recovery (85.45%) and rapid magnetic separation (within 10 s). Moreover, the as-prepared IMAC nanoparticle provides effective enrichment of phosphopeptides from real samples (human serum and nonfat milk), showing great potential as a tool for the detection and identification of low-abundance phosphopeptides in biological samples.
Co-reporter:Qianhao Min, Ren'an Wu, Liang Zhao, Hongqiang Qin, Mingliang Ye, Jun-Jie Zhu and Hanfa Zou
Chemical Communications 2010 - vol. 46(Issue 33) pp:NaN6146-6146
Publication Date(Web):2010/07/27
DOI:10.1039/C0CC00619J
In this study, the concept of size-selective proteolysis was first described by using the mesoporous silica-based trypsin nanoreactor. For analysis of a complex protein sample, low-MW proteins were preferentially digested for identification while high-MW proteins were excluded from digestion.
Co-reporter:Hongqiang Qin, Fangjun Wang, Yi Zhang, Zhengyan Hu, Chunxia Song, Ren'an Wu, Mingliang Ye and Hanfa Zou
Chemical Communications 2012 - vol. 48(Issue 50) pp:NaN6267-6267
Publication Date(Web):2012/05/01
DOI:10.1039/C2CC31705B
Highly site-selective dimethyl labeling of N-terminus of peptides has been obtained by adjusting the acidic strength of the reaction solution. And this selective labeling strategy combined with the use of different isotope formaldehyde reagents has been successfully used in the proteome quantification by using the isobaric peptide cross-sequence labeling method, which would play increasingly important roles in the clinical diagnosis, especially in the discovery of biomarkers for diseases.
Co-reporter:Yu Zhang, Chen Chen, Hongqiang Qin, Ren'an Wu and Hanfa Zou
Chemical Communications 2010 - vol. 46(Issue 13) pp:NaN2273-2273
Publication Date(Web):2010/01/21
DOI:10.1039/B921331G
Ti-hexagonal mesoporous silica (Ti-HMS) with high titanium content has been synthesized. The Ti-HMS has been applied as a potential adsorbent for the selective capture of phosphopeptides, due to the strong affinity interaction between the incorporated titanium in the framework and the phosphoryl groups of phosphopeptides.
Co-reporter:Zhichao Xiong, Hongqiang Qin, Hao Wan, Guang Huang, Zhang Zhang, Jing Dong, Lingyi Zhang, Weibing Zhang and Hanfa Zou
Chemical Communications 2013 - vol. 49(Issue 81) pp:NaN9286-9286
Publication Date(Web):2013/08/29
DOI:10.1039/C3CC45008B
Magnetic nanoparticles (MNPs) coated with multilayer polysaccharide shells have been fabricated using a layer-by-layer approach via the alternate deposition of hyaluronan (HA) and chitosan (CS) onto the surface, and the hydrophilic materials were utilized for effective and selective enrichment of glycopeptides in biological samples.
Co-reporter:Hongqiang Qin, Zhengyan Hu, Fangjun Wang, Yi Zhang, Liang Zhao, Guiju Xu, Ren'an Wu and Hanfa Zou
Chemical Communications 2013 - vol. 49(Issue 45) pp:NaN5164-5164
Publication Date(Web):2013/04/15
DOI:10.1039/C3CC41479E
Silica–carbon composite nanoparticles (NP-MCM-C) with uniform shapes and highly ordered mesoporous structures are directly prepared by using template polymers as the carbon source. And, taking advantage of the size exclusion effect of the mesopores to proteins and the specific interaction between carbon and oligosaccharides, the prepared nanoparticles are utilized to enrich N-linked glycans from complex biological samples with high selectivity and efficiency.
Co-reporter:Jinan Li, Fangjun Wang, Jing Liu, Zhichao Xiong, Guang Huang, Hao Wan, Zheyi Liu, Kai Cheng and Hanfa Zou
Chemical Communications 2015 - vol. 51(Issue 19) pp:NaN4096-4096
Publication Date(Web):2015/02/03
DOI:10.1039/C5CC00187K
Magnetic nanoparticles functionalized with maltosylated glycopeptide dendrimers were prepared via azide-alkynyl click reaction. The functionalized magnetic nanoparticles exhibited high hydrophilicity and good efficiency in glycopeptide enrichment by HILIC.
Co-reporter:Yixin Zhang, Yanbo Pan, Wujun Liu, Yongjin J. Zhou, Keyun Wang, Lei Wang, Muhammad Sohail, Mingliang Ye, Hanfa Zou and Zongbao K. Zhao
Chemical Communications 2016 - vol. 52(Issue 40) pp:NaN6692-6692
Publication Date(Web):2016/04/14
DOI:10.1039/C6CC02386J
An approach combining in vivo protein allylation, chemical tagging and affinity enrichment was devised to capture protein methylation candidates in yeast S. cerevisiae. The study identified 167 hits, covering many proteins with known methylation events on different types of amino acid residues.
Co-reporter:
Analytical Methods (2009-Present) 2013 - vol. 5(Issue 12) pp:
Publication Date(Web):
DOI:10.1039/C3AY00064H
The titanium silicalite-1 (TS-1) with post-treatment of chemical selective desilication (T-TS-1) has been synthesized, characterized and applied as a potential adsorbent for selective capture of phosphopeptides from complex biological samples prior to mass spectrometry analysis. A T-TS-1 material-based successive phosphopeptide enrichment strategy has also been established for real biological sample analysis, which exhibited high enrichment selectivity and is comparable to existing materials.
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