The launch of the Chromosome-Centric Human Proteome Project provides an opportunity to gain insight into the human proteome. The Chinese Human Chromosome Proteome Consortium has initiated proteomic exploration of protein-encoding genes on human chromosomes 1, 8, and 20. Collaboration within the consortium has generated a comprehensive proteome data set using normal and carcinomatous tissues from human liver, stomach, and colon and 13 cell lines originating in these organs. We identified 12,101 proteins (59.8% coverage against Swiss-Prot human entries) with a protein false discovery rate of less than 1%. On chromosome 1, 1,252 proteins mapping to 1,227 genes, representing 60.9% of Swiss-Prot entries, were identified; however, 805 proteins remain unidentified, suggesting that analysis of more diverse samples using more advanced proteomic technologies is required. Genes encoding the unidentified proteins were concentrated in seven blocks, located at p36, q12-21, and q42-44, partly consistent with correlation of these blocks with cancers of the liver, stomach, and colon. Combined transcriptome, proteome, and cofunctionality analyses confirmed 23 coexpression clusters containing 165 genes. Biological information, including chromosome structure, GC content, and protein coexpression pattern was analyzed using multilayered, circular visualization and tabular visualization. Details of data analysis and updates are available in the Chinese Chromosome-Centric Human Proteome Database (http://proteomeview.hupo.org.cn/chromosome/).Keywords: C-HPP; chromosome; coexpression; data visualization; proteome;

A new strategy using tandem 18O stable isotope labeling (TOSIL) to quantify the N-glycosylation site occupancy is reported. Three heavy oxygen atoms are introduced into N-glycosylated peptides: two 18O atoms are incorporated into the carboxyl terminal of all peptides during a tryptic digestion, and the third 18O atom is incorporated into the N-glycosylation site of asparagines-linked sugar chains specifically via a N-glycosidase F (PNGase F)-mediated hydrolysis. Comparing samples treated in H218O and samples treated in H216O, a unique mass shift of 6 Da can be shown for N-glycosylated peptide with single glycosylation site, which could be easily distinguished from those nonglycosite peptide pairs with a mass difference of 4 Da only. The relative quantities of N-glycosylated and its parent protein-levels were obtained simultaneous by measuring the intensity ratios of 18O/16O for glycosylated (6 Da) and for nonglycosylated (4 Da) peptides, respectively. Thus, a comparison of these two ratios can be utilized to evaluate the changes of occupancy of N-glycosylation at specific sites between healthy and diseased individuals. The TOSIL approach yielded good linearity in quantitative response within 10-fold dynamic range with the correlation coefficient r2 > 0.99. The standard deviation (SD) ranged from 0.06 to 0.21, for four glycopeptides from two model glycoproteins. Furthermore, serums from a patient with ovarian cancer and healthy individual were used as test examples to validate the novel TOSIL method. A total of 86 N-glycosylation sites were quantified and N-glycosylation levels of 56 glycopeptides showed significant changes. Most changes in N-glycosylation at specific sites have the same trends as those of protein expression levels; however, the occupancies of three N-glycosylation sites were significantly changed with no change in proteins levels.Keywords: 18O-labeling; Mass spectrometry; N-glycosylation; Ovarian cancer; Quantitative proteomics;

•SIRT5 desuccinylates and activates PKM2•Lys311 is a key succinylated site in the regulation of PKM2 activity•Sirt5 blocks IL-1β production in LPS-activated macrophages by regulating PKM2•SIRT5 plays an important role in inhibiting inflammationLPS-activated macrophages undergo a metabolic shift from dependence on mitochondria-produced ATP to reliance on aerobic glycolysis, where PKM2 is a critical determinant. Here, we show that PKM2 is a physiological substrate of SIRT5 and that SIRT5-regulated hypersuccinylation inhibits the pyruvate kinase activity of PKM2 by promoting its tetramer-to-dimer transition. Moreover, a succinylation-mimetic PKM2 K311E mutation promotes nuclear accumulation and increases protein kinase activity. Furthermore, we show that SIRT5-dependent succinylation promotes PKM2 entry into nucleus, where a complex of PKM2-HIF1α is formed at the promoter of IL-1β gene in LPS-stimulated macrophages. Activation of PKM2 using TEPP-46 attenuates Sirt5-deficiency-mediated IL-1β upregulation in LPS-stimulated macrophages. Finally, we find that Sirt5-deficient mice are more susceptible to DSS-induced colitis, which is associated with Sirt5 deficiency prompted PKM2 hypersuccinylation and boosted IL-1β production. In conclusion, our findings reveal a mechanism by which SIRT5 suppresses the pro-inflammatory response in macrophages at least in part by regulating PKM2 succinylation, activity, and function.Download high-res image (321KB)Download full-size image

•A novel 3-D floor-like ordered mesoporous carbon functionalized graphene composites FLOMC-GO have been synthesized.•The content of carbon reached 80% with highly graphitized.•A great performance has been shown that the material could capture lots of N-linked glycans.•The FLOMC-GO could efficiently size-exclude proteins and enrich the N-linked glycans released from human serum.Novel 3-D floor-like ordered mesoporous carbon functionalized graphene composites, FLOMC-GO, with high graphitized carbon contents were successfully synthesized using a soft template method. The one-pot sol-gel method was employed to prepare the silica soft template. Then, the sandwich-like composites were further combined together to form a 3-D structure through pre-carbonization and carbonization. During these procedures, the sulphonyl and sulfide bridges were formed by cross-linking processes to connect the phenyl rings. The prepared FLOMC-GO was confirmed to have a large pore volume (1.03 cm3 g−1), high BET surface area (544.99 m2 g−1) and well-ordered mesoporous structure with a narrow pore-size concentrated at 3.74 nm. The content of carbon reached 80% and was highly graphitized. By taking advantage of the interactions between carbon and glycans, FLOMC-GO was utilized to enrich N-linked glycans from OVA and human serum. As expected, excellent size-exclusion was found during the enrichment of N-glycans released from OVA, and 25 N-linked glycans were identified. The intensity of glycans enriched by FLOMC-GO was 7 times to the result of active carbon, while the ratio of OVA digestion to BSA interfering proteins increased to 1:50. Additionally, 31 N-linked glycans in total were enriched from human serum. The relatively easy synthesis as well as ability to enrich N-linked glycans with high selectivity and efficiency makes FLOMC-GO a promising adsorbent material for the discovery of human serum biomarkers for disease diagnosis.Download high-res image (176KB)Download full-size image

The coexisting post-translational modifications (PTMs) on histone H3 N-terminal tails were known to crosstalk between each other, indicating their interdependency in the epigenetic regulation pathways. H3K36 methylation, an important activating mark, was recently reported to antagonize with PRC2-mediated H3K27 methylation with possible crosstalk mechanism during transcription regulation process.1 On the basis of our previous studies, we further integrated RP/HILIC liquid chromatography with MRM mass spectrometry to quantify histone PTMs from various mouse organs, especially the combinatorial K27/K36 marks for all three major histone H3 variants. Despite their subtle difference in physicochemical properties, we successfully obtained decent separation and high detection sensitivity for both histone H3.3 specific peptides and histone H3.1/3.2 specific peptides. In addition, the overall abundance of H3.3 can be quantified simultaneously. We applied this method to investigate the pattern of the combinatorial K27/K36 marks for all three major histone H3 variants across five mouse organs. Intriguing distribution differences were observed not only between different H3 variants but also between different organs. Our data shed the new insights into histone codes functions in epigenetic regulation during cell differentiation and developmental process.

•A rapid and green chemometric-enhanced full scan mode of LC–MS method was developed.•ATLD was employed to solve the overlapped peaks and uncalibrated interferences.•ATLD-LC–MS was applied to determine six SOADs in health teas and human plasma samples.•The proposed method proved to be fast, low-cost, high sensitivity and simple implementation.•This methodology conformed to the highly recommended green analytical chemistry principles.In this work, a rapid chemometrics-enhanced liquid chromatography–single stage mass spectrometry (LC–MS) was developed for the simultaneous green determination of six co-eluted sulfonylurea-type oral antidiabetic agents (SOADs) in health teas and human plasma samples. Shortening the chromatographic separation time and reducing the cost per analysis were achieved via using full scan mode of LC–MS under simple isocratic elution condition followed by an environment-friendly “mathematical separation” strategy. The problem of the complete separation of target analytes from each other and/or from the uncalibrated interferences in complex matrices was resolved by alternating trilinear decomposition (ATLD) method as a posteriori. Satisfactory qualitative and quantitative results were obtained even in the existence of unknown interferences and the “second-order advantage” was fully exploited. The average spiked recoveries for all target analytes were between 81.6% and 110.1% with standard deviations less than 7.7%. It was demonstrated that the proposed strategy could be promisingly used for green resolution and determination of co-eluted multi-analytes of interest in complex samples while avoiding elaborate sample pretreatment steps and complicated experimental conditions as well as more sophisticated high-cost instrumentations.

•A facile and rapid platform for both visual detection and Raman quantitation.•High sensitivity and selectivity via the formation of NPs self-assembled interface.•Long-term stability of reaction products for at least three weeks.•Capability for determination of large scale samples.•Successful application of real sample.One of the main problems of the nanoparticle dispersion state change (e.g. from dispersion to aggregation) based surface-enhanced Raman scattering (SERS) detection is that the dynamic process of such in-solution reactions is always uncontrollable. This leads to poor reproducibility from a narrow time window of all such strategies, and finally great difference between the data from the diverse methods, and even between various sample batches. To address such problem, a facile, rapid SERS quantification protocol has been developed relying on target induced nanoparticle self-assembly at oil/water interfaces for copper ions analysis. In response to copper, the core-molecule-shell (CMS) nanoparticles spontaneously migrate to the interface and are assembled into densely packed arrays generating strong plasmonic coupling, which enables stable, sensitive and selective Raman quantitation, as well as visual detection. Also, this strategy shows capability for determination of large scale samples as the products can be stable for at least three weeks, and has been successfully applied to real sample detection. The developed Target Induced Nanoparticle Self-Assembled Interface (TINSAI) can be employed to both visual test and Raman quantitative detection, which would provide a platform for on-site screening as well as high-throughput detection with high sensitivity and selectivity.

A general and effective enzymatic labeling method, termed glycan reducing end dual isotopic labeling (GREDIL), was developed for mass spectrometry-based quantitative N-glycomics.

•In-gel NHS is comparable with the well-established method for histone derivatization in solution.•Suitable for analysis of histone PTMs in highly complex sample without fussy purification.•Sample processed with In-gel NHS method can be quantified with CV less than 15%.Post-translational modifications (PTMs) on histone are highly correlated with genetic and epigenetic regulation of gene expression from chromatin. Mass spectrometry (MS) has developed to be an optimal tool for the identification and quantification of histone PTMs. Derivatization of histones with chemicals such as propionic anhydride, N-hydroxysuccinimide ester (NHS-propionate) has been widely used in histone PTMs analysis in bottom-up MS strategy, which requires high purity for histone samples. However, biological samples are not always prepared with high purity, containing detergents or other interferences in most cases. As an alternative approach, an adaptation of in gel derivatization method, termed In-gel NHS, is utilized for a broader application in histone PTMs analysis and it is shown to be a more time-saving preparation method.The proposed method was optimized for a better derivatization efficiency and displayed high reproducibility, indicating quantification of histone PTMs based on In-gel NHS was achievable. Without any traditional fussy histone purification procedures, we succeeded to quantitatively profile the histone PTMs from Arabidopsis with selective knock down of CLF (clf-29) and the original parental (col) with In-gel NHS method in a rapid way, which indicated the high specificity of CLF on H3K27me3 in Arabidopsis. In-gel NHS quantification results also suggest distinctive histone modification patterns in plants, which is invaluable foundation for future studies on histone modifications in plants.

•PDS/ATLD is firstly proposed for transferring LC–MS signals measured on different days.•ATLD method is employed to solve the overlapped peaks and potential interferences.•PDS is carried out to overcome the signal change over time once the ATLD model is established.•Results from the PDS/ATLD strategy are comparable to those from the complete recalibration.•This methodology is in accordance with the recommended green analytical chemistry principles.The application of calibration transfer methods has been successful in combination with near-infrared spectroscopy or other tools for prediction of chemical composition. One of the developed methods that can provide accurate performances is the piecewise direct standardization (PDS) method, which in this paper is firstly applied to transfer from one day to another the second-order calibration model based on alternating trilinear decomposition (ATLD) method built for the interference-free resolution and determination of multi-analytes in complex systems by liquid chromatography–mass spectrometry (LC–MS) in full scan mode. This is an example of LC–MS analysis in which interferences have been found, making necessary the use of second-order calibration because of its capacity for modeling this phenomenon, which implies analytes of interest can be resolved and quantified even in the presence of overlapped peaks and unknown interferences. Once the second-order calibration model based on ATLD method was built, the calibration transfer was conducted to compensate for the signal instability of LC–MS instrument over time. This allows one to reduce the volume of the heavy works for complete recalibration which is necessary for later accurate determinations. The root-mean-square error of prediction (RMSEP) and average recovery were used to evaluate the performances of the proposed strategy. Results showed that the number of calibration samples used on the real LC–MS data was reduced by using the PDS method from 11 to 3 while producing comparable RMSEP values and recovery values that were statistically the same (F-test, 95% confidence level) to those obtained with 11 calibration samples. This methodology is in accordance with the highly recommended green analytical chemistry principles, since it can reduce the experimental efforts and cost with regard to the use of a new calibration model built in modified conditions.

Homocysteine (hcy) is an intermediate metabolite in the metabolic pathway of cysteine and methionine. As a non-coded amino acid, hcy is not normally incorporated into protein. However, homocysteine can be recognized and activated by methionyl-tRNA synthetase (MetRs) to produce Hcy–thiolactone (HTL), which can react with the ε-amino group of a protein lysine residue. The N-hcy-linked protein carrying a free thiol group can influence protein structure and function, thus leading to severe diseases. Histone has multiple specific dynamic post-translational modifications (PTMs), especially on the N-terminal tail of histones enriched with lysine and arginine residues. In this study, we confirmed that histone H3 can be modified by HTL on lysine residue. Relative and absolute quantification methods based on mass spectrometry demonstrated the crosstalk between methylation and acetylation of H3 in response to excess HTL. Overall, our data provide novel insights into histone modifications and the regulatory mechanisms of diseases related to homocysteinylation.

Trypsin has traditionally been used for enzymatic digestion during sample preparation in shotgun proteomics. The stringent specificity of trypsin is essential for accurate protein identification and quantification. But nonspecific trypsin cleavages are often observed in LC-MS/MS-based shotgun proteomics. To explore the extent of nonspecific trypsin cleavages, a series of biological systems including a standard protein mixture, Saccharomyces cerevisiae, human serum, human cancer cell lines and mouse brain were examined. We found that nonspecific trypsin cleavages commonly occurred in various trypsin digested samples with high frequency. To control these nonspecific trypsin cleavages, we optimized fundamental parameters during sample preparation with mouse brain homogenates. These parameters included denaturing agents and protein storage time, trypsin type, enzyme-to-substrate ratio, as well as protein concentration during digestion. The optimized experimental conditions significantly decreased the ratio of partially tryptic peptides in total identifications from 28.4% to 2.8%. Furthermore, the optimized digestion protocol was applied to the study of N-glycoproteomics, and the proportions of partially tryptic peptides in enriched mixtures were also sharply reduced. Our work demonstrates the importance of controlling nonspecific trypsin cleavages in both shotgun proteomics and glycoproteomics and provides a better understanding and standardization for routine proteomics sample treatment.

Novel magnetic silica nanoparticles functionalized with layer-by-layer detonation nanodiamonds (dNDs) were prepared by coating single submicron-size magnetite particles with silica and subsequently modified with dNDs. The resulting layer-by-layer dND functionalized magnetic silica microspheres (Fe3O4@SiO2@[dND]n) exhibit a well-defined magnetite–core–silica–shell structure and possess a high content of magnetite, which endow them with high dispersibility and excellent magnetic responsibility. Meanwhile, dNDs are known for their high affinity and biocompatibility towards peptides or proteins. Thus, a novel convenient, fast and efficient pretreatment approach of low-abundance peptides or proteins was successfully established with Fe3O4@SiO2@[dND]n microspheres. The signal intensity of low-abundance peptides was improved by at least two to three orders of magnitude in mass spectrometry analysis. The novel microsphere also showed good tolerance to salt. Even with a high concentration of salt, peptides or proteins could be isolated effectively from samples. Therefore, the convenient and efficient enrichment process of this novel layer-by-layer dND-functionalized microsphere makes it a promising candidate for isolation of protein in a large volume of culture supernatant for secretome analysis. In the application of Fe3O4@SiO2@[dND]n in the secretome of hepatoma cells, 1473 proteins were identified and covered a broad range of pI and molecular weight, including 377 low molecular weight proteins.

As a key post-translational modification mechanism, protein acetylation plays critical roles in regulating and/or coordinating cell metabolism. Acetylation is a prevalent modification process in enzymes. Protein acetylation modification occurs in sub-stoichiometric amounts; therefore extracting biologically meaningful information from these acetylation sites requires an adaptable, sensitive, specific, and robust method for their quantification. In this work, we combine immunoassays and multiple reaction monitoring-mass spectrometry (MRM-MS) technology to develop an absolute quantification for acetylation modification. With this hybrid method, we quantified the acetylation level of metabolic enzymes, which could demonstrate the regulatory mechanisms of the studied enzymes. The development of this quantitative workflow is a pivotal step for advancing our knowledge and understanding of the regulatory effects of protein acetylation in physiology and pathophysiology.

SDS-PAGE and high-pH RPLC are commonly used fractionation strategies in proteomics research. A comparative investigation of these two strategies would be meaningful to thoroughly understand their respective features. Here, we systematically compared the two methods by trying 4 SDS-PAGE/RPLC and 3 high-/low-pH RPLC different workflows for a higher sensitivity in protein identification. Totally 9793 proteins were identified in HepG2 cells, with 8581 proteins identified by high-/low-pH RPLC workflows and 7933 by SDS-PAGE/RPLC workflows. The results demonstrate that using high-pH RPLC in the first dimensional separation would favour a high-throughput proteome analysis but choosing SDS-PAGE could yield much better peptide coverage. We found that the SDS-PAGE fractionation method benefits the neutral pI peptides. We also analyzed unexpected modifications caused by the two strategies. Our results suggest that more pre-fractionation benefits protein identifications in both strategies and pooling of gel pieces according to their grey values increased the identification efficiency in SDS-PAGE/RPLC workflows.

PNGase F-catalyzed glycosylation site 18O-labeling is a widely used method for glycoprotein quantitation owing to its efficiency and simplicity. However, PNGase F-catalyzed glycan 18O-labeling, which offers advantages for glycomics, has not yet been developed. In this study, PNGase F-mediated incorporation of 18O into glycans during the N-glycan release from glycoproteins by PNGase F was finally realized, named as PCGOL (PNGase F-catalyzed glycan 18O-labeling), which offers a potential strategy for relative glycan quantitation. This new method showed good linearity and high reproducibility within at least 2 orders of magnitude in the dynamic range. Furthermore, PCGOL combined with our previously developed TOSIL method (tandem 18O stable isotope labeling for N-glycoproteome quantitation) can be used for comprehensive N-glycosylation quantification, achieving simultaneous quantification of glycans, glycopeptides and glycoproteins in a single workflow, which was also used to analyze glycosylation changes in immunoglobulin G (IgG) associated with hepatocellular carcinoma in the present work.

HeLa cell line, which was derived from cervical carcinoma, provides an idea platform to study both the integration of human papillomavirus and the massive mutations occurring on the cancer cell genome. Proteogenomics is a field with the intersection of proteomics and genomics to perform gene annotation and identify gene mutation. In this work, we first identified the SNV/INDEL, structural variation (SV), and virus infection/integration events from RNA-Seq data of HeLa cell line; then, by applying proteogenomics strategy, we were able to detect some of the genomic events with the tandem mass spectrometry (MS/MS) data from the same sample. Furthermore, some of the mutated peptides were experimentally validated using multiple reaction monitoring technology. The integrated analysis of the RNA-Seq and MS/MS data not only renders the discovery of HeLa cell genome variations more credible but also illustrates a practical workflow for protein-coding mutation discovery in cancer-related studies.

The dispersive effect of polyacrylate amine (NH4PAA) upon suspensions of silicon nitride (Si3N4) is considered. Slurry introduction for axial inductively coupled plasma optical emission spectrometry determination of Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni and Ti in powdered Si3N4 has also been investigated. Characterization by zeta potential measurement and slurry stability measurements shows that NH4PAA is a good dispersant for the preparation of Si3N4 suspensions. The optimal concentration of NH4PAA was 0.8 wt% for μm-sized Si3N4 and 1.2 wt% for nm-sized Si3N4. The analytical results obtained using nm- and μm-sized Si3N4 by slurry introduction were in good accordance with results obtained by the high-pressure acid deposition method, thus verifying that calibration curves could be established using aqueous standards. By introducing a low amount of contamination in the sample preparation, an accurate result is obtained for the detection of contamination-risk elements such as K and Na, which cannot be accurately determined by the high-pressure acid deposition method. The limits of detection, which are in the range of 8–250 ng g−1, were shown to be superior to those of the conventional nebulization technique with ICP-OES or ICP-MS.

•Site-specific O-glycosylation of recombinant human osteopontin (OPN) was analyzed.•28 O-glycopeptides carrying highly heterogeneous O-glycans were detected.•Some important carbohydrate epitopes were revealed.•26 phosphorylation sites were identified, including a novel one (Y209).Osteopontin (OPN) plays a key role in multiple physiological and pathological processes such as cytokine production, mineralization, inflammation, immune responses, and tumorigenesis. Post-translational modifications (PTMs) of OPN significantly affect its structure and biological properties; however, site-specific characterization of O-glycosylation in human OPN has not been reported. In this work, we profiled the overall glycan pattern of human recombinant OPN using a lectin array and completed detailed structural analysis of O-glycopeptides by mass spectrometry (MS). We detected 28 O-glycopeptides from 7 O-glycosylation regions of human OPN, occupied by highly heterogeneous O-glycans. These O-glycans carried, in part, functionally relevant epitopes such as T antigens (Galβ1-3GalNAcα1-), sialyl-Tn antigens, sialyl-T antigens, and sialyl-Lex/a antigens [Neuα2-3Galβ1-4(Fucα1-3)GlcNAc/Neuα2-3Galβ1-3(Fucα1-4)GlcNAc]. MS3 spectra of the generated O-glycopeptides showed cleavages of the peptide backbone and provided essential information on the peptide sequence. Furthermore, 26 phosphorylation sites were identified by reverse-phase liquid chromatography–tandem mass spectrometry (RPLC–MS/MS), including a novel one (Y209). We provide a detailed, site-specific structural characterization of O-glycosylation and identify the phosphorylation sites of OPN. These data lay the foundation for further research into the role of oligosaccharides and phosphorylation of recombinant human OPN. This article is part of a Special Issue entitled: Medical Proteomics.

•A smart strategy that combines three-way LC–MS data with ATLD method was developed.•This strategy was employed to determine ten β-blockers in human urine and plasma samples.•The results indicated the proposed strategy can solve co-elution problems in quantitative LC–MS.•This strategy proved to be a powerful tool to enhance the selectivity and sensitivity of LC–MS.•The performances of the proposed strategy were compared with those of MRM method.β-blockers are the first-line therapeutic agents for treating cardiovascular diseases and also a class of prohibited substances in athletic competitions. In this work, a smart strategy that combines three-way liquid chromatography–mass spectrometry (LC–MS) data with second-order calibration method based on alternating trilinear decomposition (ATLD) algorithm was developed for simultaneous determination of ten β-blockers in human urine and plasma samples. This flexible strategy proved to be a useful tool to solve the problems of overlapped peaks and uncalibrated interferences encountered in quantitative LC–MS, and made the multi-targeted interference-free qualitative and quantitative analysis of β-blockers in complex matrices possible. The limits of detection were in the range of 2.0 × 10−5–6.2 × 10−3 μg mL−1, and the average recoveries were between 90 and 110% with standard deviations and average relative prediction errors less than 10%, indicating that the strategy could provide satisfactory prediction results for ten β-blockers in human urine and plasma samples only using liquid chromatography hyphenated single–quadrupole mass spectrometer in full scan mode. To further confirm the feasibility and reliability of the proposed method, the same batch samples were analyzed by multiple reaction monitoring (MRM) method. T-test demonstrated that there are no significant differences between the prediction results of the two methods. Considering the advantages of fast, low-cost, high sensitivity, and no need of complicated chromatographic and tandem mass spectrometric conditions optimization, the proposed strategy is expected to be extended as an attractive alternative method to quantify analyte(s) of interest in complex systems such as cells, biological fluids, food, environment, pharmaceuticals and other complex samples.

The CXCL12/CXCR4 axis has been posited widely to have significant roles in many primary tumors and metastases. It is known that CXCR7 can also be engaged by CXCL12, but the exact function of CXCR7 is controversial. This prompted us to investigate the expression, specific function and signal transduction of CXCR7 in hepatocellular carcinoma (HCC). In this study, CXCR7 and CXCR4 were differentially expressed in nine cell lines of HCC, and that elevated expression of both CXCR7 and CXCL4 were correlated with highly metastatic ability of HCC cells. Moreover, CXCR7 expression was significantly upregulated in metastatic HCC samples compared with the non-metastatic ones by staining of high-density tissue microarrays constructed from a cohort of 48 human HCC specimens. CXCR7 overexpression enhanced cell growth and invasiveness in vitro, and tumorigenicity and lung metastasis in vivo. By contrast, CXCR7 stable knockdown markedly reduced these malignant behaviors. In addition, it was observed that alterations in CXCR7 expression were positively correlated with the phosphorylation levels of mitogen-activated protein kinase (MAPK) pathway proteins. Targeting extracellular regulated kinase pathway by using U0126 inhibitor or using CCX771, a selective CXCR7 antagonist, drastically reduced CXCR7-mediated cell proliferation. Importantly, by using human biotin-based antibody arrays, several differentially expressed proteins were identified in CXCR7-overexpression and depletion groups. Comparative analysis indicated that upstream regulators including TP53 and IL-6 were involved in CXCR7 signal transduction. CXCR7 expression was further proved to regulate expression of vascular endothelial growth factor A and galectin-3, which may contribute to tumor angiogenesis and invasiveness. Consequently, elevated expression of CXCR7 contributes to HCC growth and invasiveness via activation of MAPK and angiogenesis signaling pathways. Targeting CXCR7 may prevent metastasis and provide a potential therapeutic strategy for HCC.

To estimate the potential of the state-of-the-art proteomics technologies on full coverage of the encoding gene products, the Chinese Human Chromosome Proteome Consortium (CCPC) applied a multiomics strategy to systematically analyze the transciptome, translatome, and proteome of the same cultured hepatoma cells with varied metastatic potential qualitatively and quantitatively. The results provide a global view of gene expression profiles. The 9064 identified high confident proteins covered 50.2% of all gene products in the translatome. Those proteins with function of adhesion, development, reproduction, and so on are low abundant in transcriptome and translatome but absent in proteome. Taking the translatome as the background of protein expression, we found that the protein abundance plays a decisive role and hydrophobicity has a greater influence than molecular weight and isoelectric point on protein detectability. Thus, the enrichment strategy used for low-abundant transcription factors helped to identify missing proteins. In addition, those peptides with single amino acid polymorphisms played a significant role for the disease research, although they might negligibly contribute to new protein identification. The proteome raw and metadata of proteome were collected using the iProX submission system and submitted to ProteomeXchange (PXD000529, PXD000533, and PXD000535). All detailed information in this study can be accessed from the Chinese Chromosome-Centric Human Proteome Database.

To completely annotate the human genome, the task of identifying and characterizing proteins that currently lack mass spectrometry (MS) evidence is inevitable and urgent. In this study, as the first effort to screen missing proteins in large scale, we developed an approach based on SDS-PAGE followed by liquid chromatography–multiple reaction monitoring (LC-MRM), for screening of those missing proteins with only a single peptide hit in the previous liver proteome data set. Proteins extracted from normal human liver were separated in SDS-PAGE and digested in split gel slice, and the resulting digests were then subjected to LC-schedule MRM analysis. The MRM assays were developed through synthesized crude peptides for target peptides. In total, the expressions of 57 target proteins were confirmed from 185 MRM assays in normal human liver tissues. Among the proved 57 one-hit wonders, 50 proteins are of the minimally redundant set in the PeptideAtlas database, 7 proteins even have none MS-based information previously in various biological processes. We conclude that our SDS-PAGE-MRM workflow can be a powerful approach to screen missing or poorly characterized proteins in different samples and to provide their quantity if detected. The MRM raw data have been uploaded to ISB/SRM Atlas/PASSEL (PXD000648).

Study of site-specific N-glycosylation in complex sample remains a huge analytical challenge because protein glycosylation is structurally diverse in post-translational modifications, resulting in an intricacy of N-glycopeptides. Here we have developed a novel approach for high-throughput N-glycopeptide profiling based on a network-centric algorithm for deciphering glycan fragmentation in mass spectrometry. We performed an extensive validation and a high-throughput N-glycosylation study on serum and identified thousands of N-glycopeptide spectra with high confidence. The results revealed a similar level of glycan microheterogeneity to that of conventional glycomics approach on individual proteins and provided the unique in-depth site-specific information that could only be studied through glycopeptide profiling.

Slurry nebulization in plasmas has advantages of simplicity, high speed, low cost, minimized analyte loss, and low risk of sample contamination, but it has not been very widely adopted. However, the study of advanced ceramic materials has recently renewed enthusiasm for this technique. In this paper, the current state of research on slurry nebulization in plasmas for the analysis of advanced materials is thoroughly surveyed. Sample preparation, sample characterization, and modifications of the instrumentation necessary for slurry nebulization are reviewed, along with calibration procedures and studies on fundamental issues. Finally, the applications of this method to a variety of advanced materials are summarized, and the outlook for this method is discussed.

The liver is an important organ and is the biggest digestive gland in the human body. The establishment of a proteome database of the human liver would provide useful information for human liver disease treatment. In order to maximize protein identification and to compare different analyses, multiple technical routes were used for proteome profiling of French liver samples. Five strategies, including two two-dimensional electrophoresis (2DE) methods and three multi-dimensional liquid chromatography (MDLC) methods were evaluated. By using these five routes, 1627 unique proteins were finally identified. Various bioinformatics analyses focused on physicochemical properties and functional classification were used to examine the characteristics of the protein expression profile. Furthermore, comparison of these data with an existing liver expression profile provided by UNIGENE and UNIPROT allowed us to investigate the identification efficiency of this dataset. The different technical methods were evaluated and compared to make a model of the organ proteome profile.

Lectin microarrays are increasingly used as screening tools with a high potential for glycobiology analysis, providing a high-sensitivity profiling of complex glycan features in a short time, without the need for the release of glycan. The application of the technology for the rapid differential glycan profiling of target samples resulted in the discovery of new glycan-related biomarkers for disease detection and particular types of glycobiology studies. Additionally, lectin microarrays might offer many opportunities for further innovation, and their range of applications in the life sciences promises to be greatly expanded. Herein, we describe the advances in the fabrication and detection of lectin microarray technologies and their application, which herald even more exciting breakthroughs in the coming decade.

To explore the proteomic changes of placental trophoblastic cells in preeclampsia–eclampsia (PE), placental trophoblastic cells from normally pregnant women and women with hypertension during gestational period were prepared by laser capture microdissection (LCM), and proteins isolated from these cells were subjected to labeling and proteolysis with isotope-coded affinity tag reagent. A qualitative and quantitative analysis of the proteome expression of placental trophoblastic cells was made using two-dimensional liquid chromatography tandem mass spectrometry (2D LC–MS/MS). A total of 831 proteins in placental trophoblastic cells were identified by combined use of LCM technique and 2D LC–MS/MS. The result was superior to that of conventional two-dimensional electrophoresis method. There were marked differences in 169 proteins of placental trophoblastic cells between normally pregnant women and women with PE. Of 70 (41.4 %) proteins with more than twofold differences, 31 proteins were down-regulated, and 39 were up-regulated in placental trophoblastic cells of the woman with PE. Laminin expression in placenta trophoblastic cells of women with PE was significantly down-regulated as confirmed by Western blot analysis. These findings provide insights into the proteomic changes in placental trophoblastic cells in response to PE and may identify novel protein targets associated with the pathogenesis of PE.

Snake venom is a complex cocktail including a variety of biological active proteins and proteinaceous components, which have considerable medical and pharmacological importance. N-Glycosylation is widely implicated as a common modification in numerous venom proteins and impacts the in vivo venomic functions. However, systematic survey of N-glycome and N-glycoproteome on snake venoms has not been undertaken. In this study, employing combination of N-glycomics and N-glycoproteomics strategies, we explored the N-glycosylation including both N-glycoproteins and N-glyco-chains in three venoms from Agkistrodon blomhoffii, Naja naja atra Cantor and Vipera russelii siamensis Smith, respectively, which are amongst the most abundant venomous snakes in Asia. As a result, numbers of N-glycoproteins and N-glycans were identified. However, the overlaps of N-glycoproteins and N-glycans of the three venoms were small. Thus, the exploration results of N-glycome and N-glycoproteome indicate that N-glycosylation increases the complexity and variety of the three venoms. Our research provided some new horizons for the comprehensive understanding of venoms variation, which is helpful for the basic venom research as well as the management of snake envenomation.

High-throughput quantification with label-free methods has received considerable attention in electrospray ionization (ESI)-mass spectrometry (MS), but the manner by which MS signals respond to peptide concentration remains unclear in proteomics. We developed a new mathematical formula to describe the intrinsic log-log relationship between the MS intensity response and peptide concentration in an analytical ESI process. Experimental results showed that the calibration curve is fairly fit to the log-log formula with a linear dynamic range of approximate four to five orders of magnitude. However, we found that the ionization of analytical peptides can be severely suppressed by coexisting matrix peptides, such that the calibration curve can be poorly leveled off on both ends. Our study suggests that the interferences from coexisting matrix peptides should be reduced in the ESI process to use the log-log calibration curve successfully for the high-throughput quantification.

A facile online enrichment protocol has been proposed based on microfluidic droplets acting as an interface between a liquid chromatography separation system and detection systems of ESI-MS/MS and laser-induced fluorescence. Low-abundance species were successfully concentrated and analyzed in this system via droplet shrinkage. The proposed platform significantly increased the enrichment efficiency and detection sensitivity with reduced sample handling steps, short analysis time, and no cross-contamination. The presented system is universal, shows no discrimination, and is easily coupled with other separation and detection approaches.

Secretomics is receiving more and more considerable attention due to the key roles of secreted proteins in cancer. Most of the potential biomarkers for clinical diagnosis and treatment of cancer are secreted proteins. However, the low concentration of secreted proteins and contaminants released from dead cells are a great challenge to secretomic profiling studies. Although some bioinformatics tools such as SecretomeP and SignalP can help to annotate or predict secreted proteins, they also cause false positive or negative rates of identification especially for nonclassical secreted proteins. Therefore, an iTRAQ based quantitative proteomics strategy was set up in this work and applied in the secretomics study of metastatic HCC cell lines. A total of 94 proteins were identified as secreted and 31 of them were newly found in our data. Compared with the known secreted proteins participating in inter-cellular signalling, most of the newly identified secreted proteins were metabolic enzymes, such as PKM2 and EHHADH, whose functions focused on the synthesis/metabolism of glucose, fatty acids and amino acids. Exploring their secretion would help to further study their bio-functions in conditioned media and the effects on the interactions of cancer cells and the microenvironment. Differences between the secretomes of the two metastatic HCC cell lines were also explored in the same experiment. This strategy showed its superiority in accurately identifying secreted proteins as well as monitoring their variation under different biological conditions.

A miniaturised reflectron time-of-flight mass spectrometer combined with an electron ionisation ion source has been developed for the analysis of gases. An entirely new helium ion removal pulsing technique in this mass spectrometer is used to achieve an improved performance for the first time. The helium carrier gas, which enters into the source along with the gaseous sample, is simultaneously ionised and then orthogonally introduced into the time-of-fight mass analyser. Once the relatively light helium ions in the ion packet become extremely close to the reflectron plate (B-plate for short in this article), a modulated pulse is instantaneously applied on the B-plate and a negative reflectron voltage is set to the B-plate and lasts for a very short period, during which all the helium ions are directly bumped into the B-plate and subsequently removed. The helium ion removal pulsing technique can efficiently avoid saturation of the micro-channel plate caused by too many helium ions. A compact and durable instrument is designed, which has a mass resolving resolution greater than 400 FWHM for online gas analysis. The technology may also be further developed to remove other ions for TOF mass spectrometry.

Here we present the novel functionalization of detonation nanodiamond (dND) with amino-phenyl boronic acid (APBA) assisted by poly-L-lysine (PL) and PEG-diglycolic acid (PEG) (dND-p-APBA). With the assistance of biocompatible poly-L-lysine and PEG, dND-p-APBA particles show good dispersibility in aqueous solution and plentiful boronic acid functional groups on the surface, which make it a novel material promising for glycoproteome. The dND-p-APBA particles demonstrate highly specific and efficient capture of glycopeptides from complex samples. In comparison with direct (or traditional) analysis, the novel method shows better specificity for glycopeptides even when non-glycopeptides are 40 times more than glycopeptides. Besides that, the detection sensitivity of glycopeptides could be increased 50 times by the novel strategy. Additionally, the recovery of glycopeptides is up to 72.2% and the glycopeptides even can be sensitively specifically enriched and detected in the presence of 1 M sodium chloride or 0.1% sodium dodecyl sulfate. Furthermore, in combination with liquid chromatography electronspray ionization mass spectrometry (LC-ESI-MS), the novel approach was successfully applied to characterize one fraction of mouse liver, in which 40 different N-glycosylation peptides within 34 unique glycoproteins have been identified. Accordingly, the usefulness of the functionalized dND might be a promising efficient and specific analytical platform in glycoproteome analysis.

•Identify the proteins involved in histological differentiation of pancreatic cancer.•Two sets of six different isobaric tags were used for the four independent groups.•Myoferlin is an independent prognostic factor for survival of PC patients.•Knockdown of MYOF alleviated malignant phenotypes of PC cell lines.AbstractHistological differentiation is a major pathological parameter associated with poor prognosis in patients with pancreatic adenocarcinoma (PAC) and the molecular signature underlying PAC differentiation may involve key proteins potentially affecting the malignant characters of PAC. We aimed to identify the proteins which could be implicated in PAC prognosis. We used isobaric tags for relative and absolute quantitation (iTRAQ) coupled with two-dimensional liquid chromatography–tandem mass spectrometry to compare protein expression in PAC tissues with different degrees of histological differentiation. A total of 1623 proteins were repeatedly identified by performing the iTRAQ-based experiments twice. Of these, 15 proteins were differentially expressed according to our defined criteria. Myoferlin (MYOF) was selected to validate the proteomic results by western blotting. Immunohistochemistry in a further 154 PAC cases revealed that myoferlin significantly correlated with the degree of histological differentiation (P = 0.004), and univariate and multivariate analyses indicated that MYOF is an independent prognostic factor for survival (hazard ratio, 1.540; 95% confidence interval, 1.061–2.234; P = 0.023) of patients with PAC after curative surgery. RNA interference-mediated knockdown of MYOF alleviated malignant phenotypes of both primary and metastatic PAC cell lines in vitro and in vivo. Thus, ITRAQ-based quantitative proteomics revealed the prognostic value of MYOF in PAC.Biological significanceOur results provide the possibility of novel strategies for pancreatic adenocarcinoma management.

•First profiling of E. granulosus adult proteome•Extended identification of protoscolex proteome reveals novel features on metabolism.•Host proteins co-purified with adult and protoscolex provide novel insights into host–parasite interplay.Cystic hydatid disease is an important zoonosis caused by Echinococcus granulosus infection. The expression profiles of its parasitic life stages and host–Echinococcus interactions remain to be elucidated. Here, we identified 157 adult and 1588 protoscolex proteins (1610 in all), including 1290 novel identifications. Paramyosins and an antigen B (AgB) were the dominant adult proteins. Dog proteins (30) identified in adults indicated diminished local inflammation caused by adult infection. The protoscolex expresses proteins that have been reported to be antigens in other parasites, such as 6-phosphofructokinase and calcineurin B. Pathway analyses suggested that E. granulosus uses both aerobic and anaerobic carbohydrate metabolisms to generate ATP. E. granulosus expresses proteins involved in synthesis and metabolism of lipids or steroids. At least 339 of 390 sheep proteins identified in protoscolex were novel identifications not seen in previous analyses. IgGs and lambda light chains were the most abundant antibody species. Sheep proteins were enriched for detoxification pathways, implying that host detoxification effects play a central role during host–parasite interactions. Our study provides valuable data on E. granulosus expression characteristics, allowing novel insights into the molecular mechanisms involved in host–parasite interactions.Biological significanceIn this study, the Echinococcus granulosus adult worm proteome was analyzed for the first time. The protein identification of E. granulosus protoscoleces was extended dramatically. We also identified the most abundant host proteins co-purified with Echinococcus. The results provide useful information pertaining to the molecular mechanisms behind host–Echinococcus interaction and Echinococcus biology. This data also increases the potential for identifying vaccine candidates and new therapeutic targets, and may aid in the development of protein probes for selective and sensitive diagnosis of echinococcosis infection. In addition, the data collected here represents a valuable proteomic resource for subsequent genome annotation.

A freezing technique protocol was proposed for coupling microchip electrophoresis with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). The microfluidic flow was frozen immediately after electrophoresis on microfluidic chip and the separated analyte molecules were kept in their zone pattern in the electrophoresis. Then, the frozen-chip was lyophilized and sent into TOF-MS instrument as a MALDI target, and the analyte molecules in the microfluidic channels were subjected to analysis by mass spectrometry. This approach could eliminate sample cross-contamination, providing a new interface for microchip electrophoresis and MALDI-MS.A freezing technique was used in coupling microfluidic chip with MALDI-TOF-MS for the first time and this protocol has been applied to peptide analyses successfully.

A segment flow protocol has been proposed for coupling nano-liquid chromatography (nano-LC) with mass spectrometry (MS) in peptide-based proteomics research. LC effluent was segmented by perfluorodecalin oil and stored as segments in sequence to carry out the following off-line MSn analysis. In this way, incomplete identification of co-eluted peptides resulting from the MS scan rate, relatively slower than the LC elution rate, was solved to some extent for on-line coupling LC-MSn analysis. The complex proteins from a sample of T. tengcongensis were successfully identified with one run using segment as the interface between nano-LC and MSn analysis, compared to the total identified proteins with six runs using routine on-line nano-LC-MSn analysis. This segment flow approach could eliminate detection loss and sample cross-contamination, providing a new method for peptide-based proteomic research, especially when the amount of sample is limited.

A versatile microreactor protocol based on microfluidic droplets has been developed for on-line protein digestion. Proteins separated by liquid chromatography are fractionated in water-in-oil droplets and digested in sequence. The microfluidic reactor acts also as an electrospray ionization emitter for mass spectrometry analysis of the peptides produced in the individual droplets. Each droplet is an enzymatic micro-reaction unit with efficient proteolysis due to rapid mixing, enhanced mass transfer and automated handling. This droplet approach eliminates sample loss, cross-contamination, non-specific absorption and memory effect. A protein mixture was successfully identified using the droplet-based micro-reactor as interface between reverse phase liquid chromatography and mass spectrometry.

A spherical liquid–liquid interface can be obtained by dispersing one liquid phase into another to form droplets, which will facilitate the two-phase reactions between the immiscible participating fluids. The phase transfer catalysts assembled at the droplet “wall” catalyze the reactions between the aqueous and organic phases. The study illustrates an interfacial synthetic approach which is ideal for the biphasic reaction by taking advantage of the droplet-based microdevice. The improved reaction efficiency can be attributed to the high surface-to-volume ratio and internal flow circulation in the droplets.

The human hepatoma 3B cell line was chosen as an experimental model for in vitro test of drug screening. The drugs included chlorophyllin and its derivatives such as fluo-chlorophyllin, sodium copper chlorophyllin, and sodium iron chlorophyllin. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-tetrazolium bromide (MTT) method was used in this study to obtain the primary screening results. The results showed that sodium iron chlorophyllin had the best LC50 value. Proteomic analysis was then performed for further investigation of the effect of sodium iron chlorophyllin addition to the Hep 3B cell line. The proteins identified from a total protein extract of Hep 3B before and after the drug addition were compared by two-dimensional-gel-electrophoresis. Then 32 three-fold differentially expressed proteins were successfully identified by MALDI-TOF-TOF-MS. There are 29 unique proteins among those identified proteins. These proteins include proliferating cell nuclear antigen (PCNA), T-complex protein, heterogeneous nuclear protein, nucleophosmin, heat shock protein A5 (HspA5) and peroxiredoxin. HspA5 is one of the proteins which are involved in protecting cancer cells against stress-induced apoptosis in cultured cells, protecting them against apoptosis through various mechanisms. Peroxiredoxin has anti-oxidant function and is related to cell proliferation, and signal transduction. It can protect the oxidation of other proteins. Peroxiredoxin has a close relationship with cancer and can eventually become a disease biomarker. This might help to develop a novel treatment method for carcinoma cancer.

The membrane glycoprotein CD82 (KAI1) has attracted increasing attention as a suppressor of cell migration, related tumor invasion, as well as metastasis. The glycosylation of CD82 has been shown to be involved in a correlative cell adhesion and motility. However, the N-glycosylation pattern of CD82 has not been described yet. In the current study, a detailed characterization of the recombinant human CD82 N-linked glycosylation pattern was conducted by employing an integrative proteomic and glycomic approach, including glycosidase and protease digestions, glycan permethylation, MS analyses, site-directed mutagenesis, and lectin blots. The results reveal three N-glycosylation sites, and further demonstrate a putative glycosylation site at Asn157 for the first time. A highly heterogeneous pattern of N-linked glycans is described, which express distinct carbohydrate epitopes, such as bisecting N-acetylglucosamine, (α-2,6) N-acetylneuraminic acid, and core fucose. These epitopes are highly associated with various biological functions, including cell adhesion and cancer metastasis, and can possibly influence the anti-cancer inhibition ability of CD82.Highlights► N-glycosylation of recombinant human CD82, a tumor-associated membrane protein, was analyzed. ► Three N-glycosylation sites were revealed, where Asn157 was demonstrated for the first time. ► 27 N-glycan structures were described for the first time. ► Some important carbohydrates including bisecting GlcNAc, NeuAc and core fucose were detected.

Parkinson’s disease (PD) is a common disease which occurs in aged people with chronic, progressive degenerative character of the central nervous system. Until now there is no effective treatment method in PD patients before they show obvious symptoms for prevention and early diagnosis. In order to find out early disease specific biomarkers, two-dimensional liquid chromatography-tandem mass spectrometry coupled with isobaric tags for relative and absolute quantification (iTRAQ) labeling was employed to quantitatively identify the differentially expressed proteins among the different disease progress types of PD. 26 proteins were differentially expressed in a total of 258 identified proteins by proteomic techniques. The expression level of eight proteins which included sero-transferrin and clusterin increased. The expression level of eighteen proteins which include complement component 4B, apolipoprotein A–I, α-2-antiplasmin and coagulation factor V decreased. Those proteins may be associated with oxidative stress, mitochondrial dysfunction, abnormal protein aggregation and inflammation. In this study, the expression level of apolipoprotein A–I decreased, particularly in the early stage of PD patients. This protein regulated not only the lipid metabolism in the central nervous system, but also influenced the deposition process of proteins which are involved in neural degenerative diseases, such as the pathogenesis of PD.

Hepatocellular carcinoma (HCC) is one of the most common and aggressive malignant tumors worldwide. The prognosis of patients with HCC still remains very dismal, mainly due to metastasis. We found that high-expression levels of AGR2 existed in metastatic HCC cell lines and patient samples. Overexpression of AGR2 was found to be correlated to the metastatic status of HCC cells, and inhibition of AGR2 by siRNA resulted in a dramatic decline in invasion abilities in metastatic cells in vitro. Overexpression of AGR2 increased the invasion of HCC cells in vitro and also in vivo with a nude mouse model. The tandem affinity purification (TAP) identified 18 AGR2-binding proteins and IPA analysis revealed that these proteins focus on MAPK and Caspase pathway. Therefore, we speculate that the overexpression of AGR2 can promote HCC metastasis, possibly by affecting MAPK and Caspase pathway through AGR2-interacting proteins.

Playing an important role in a broad range of biological and pathological processes, sialylation has been drawing wide interest. The efficient sialoglycopeptides enrichment methods are therefore attracting considerable attention. In this paper, we first compared two conventional enrichment methods, lectin and TiO2, and analyzed their characteristics. Furthermore, considering the highly negatively charged nature of sialic acids, we developed a new strategy, peptide immobilized pH gradient isoelectric focusing (IPG-IEF) assisted TiO2 chromatography (PIAT), for the highly efficient enrichment of sialoglycopeptides. In this method, peptides were first separated into 24 fractions using peptide IPG-IEF. Sialoglycopeptides were relatively concentrated in low-pH fractions of the immobilized pH strips and were captured using TiO2 chromatography. As a result, 614 N-glycosylation sites were identified in 582 sialoglycopeptides within 322 sialoglycoproteins from rat liver using PIAT. To our knowledge, this work represents one of the most comprehensive sialoglycoproteomic analyses in general and exhibits the largest database of sialoglycoproteome in rat liver currently. So the new strategy introduced here exhibits high efficiency and universality in the sialoglycopeptide enrichment, and is a powerful tool for sialoglycoproteome exploration.

A new upconversion luminescence (UCL) nanophosphors based on host matrix of cubic NaLuF4 with bright luminescence have been synthesized by a solvothermal method, facilitate the nanocrystals potential candidates for imaging in vivo, especially large-animals. The sub-20 nm NaLuF4 co-doped Yb3+ and Er3+ (Tm3+) showed about 10-fold stronger UCL emission than that of corresponding hexagonal NaYF4-based nanocrystals with a 20 nm diameter. Near-infrared to near-infrared (NIR-to-NIR) UCL emission of PAA-coated NaLuF4:20%Yb,1%Tm (PAA-LuTm) can penetrate >1.5 cm tissue of pork with high contrast. Based on super-strong UCL emission and deep penetration, PAA-LuTm as optical bioprobe has been demonstrated by in vivo UCL imaging of a normal black mouse, even rabbit with excellent signal-to-noise ratio. Furthermore, such cubic NaLuF4-based nanophosphor was applied in lymph node imaging of live Kunming mouse with rich white fur.

Stable isotopic labeling coupled with mass spectrometry analysis is a promising method of detecting quantitative variations in glycans, which may result in aberrant glycosylation in many disorders and diseases. Although various isotopic labeling methods have been used for relative glycan quantitation, enzymatic 18O labeling, which offers advantages for glycomics similar to those by protease-catalyzed 18O labeling for proteomics, has not been developed yet. In this study, endoglycosidase incorporated 18O into the N-glycan reducing end in 18O-water as N-glycans were released from glycoproteins, rendering glycan reducing-end 18O labeling (GREOL) a potential strategy for relative glycan quantitation. This proposed method provided good linearity with high reproducibility within 2 orders of magnitude in dynamic range. The ability of GREOL to quantitatively discriminate between isomeric hybrid N-glycans and complex N-glycans in glycoproteins was validated due to the distinct substrate specificities of endoglycosidases. GREOL was also used to analyze changes in human serum N-glycans associated with hepatocellular carcinoma.

Cytotrophoblast cell differentiation into syncytiotrophoblast cells is a complex and delicate process, and the mechanism needs a large number of further studies. Knockout of 14-3-3τ expression and some further investigations were performed in the syncytiotrophoblast cell formation with RNA interference (RNAi) technology. The study found that the 14-3-3τ expression level with the formation of syncytiotrophoblast cells increased. Hypoxia inhibited the formation of syncytiotrophoblast cells where 14-3-3τ expression level decreased. RNAi reduced 14-3-3τ expression, and reduced the formation of syncytiotrophoblast cells. So that 14-3-3τ is not only involved in the cell regulation, but also in the formation of syncytiotrophoblast cells. 14-3-3τ is an important regulatory factor which inhibits the formation of hypoxia.

Hepatoma-derived growth factor (HDGF) is a growth factor related to normal development and tumorigenesis; however, the mechanism of its mitogenic and angiogenic activity still remains unknown. Analysis of the HDGF interactome could be important for understanding its function and integrative mechanisms, because knowledge about HDGF interactors is very limited. In this study, through streptavidin-binding peptide (SBP) and Flag tag-based tandem affinity purification (SBP/Flag-TAP) coupled with LC-MS/MS, 106 proteins were shown to form complexes with HDGF. RNAs were also found in the HDGF complex through the SBP-tag based RNA co-immunoprecipitation (SBP-RIP) assay. Some of these interactions were confirmed by Co-IP and RT-PCR. We then found that the HATH domain was essential for HDGF interactions including protein–protein and protein–RNA interactions, and that in the absence of the HATH domain, NO-HATH could not form complex. The interactome suggests that HDGF is a multifunctional protein and participates in many cellular events, including ribosome biogenesis, RNA processing, DNA damage repair and transcriptional regulation. This new information about the HDGF interactome will further our understanding on HDGF-mediated cellular functions.Highlights► 132 proteins were identified to form complex with HDGF by SBP/FLAG-TAP coupled with LC-MS/MS. ► RNAs were also identified in the HDGF complex by SBP-tag based RNA co-immunoprecipitation assay. ► HATH domain was essential for HDGF protein–protein and protein–RNA interactions. ► The interactome study suggests HDGF is a multifunctional protein.

Systematic proteomic studying of the mechanism of hepatocellular carcinoma (HCC) metastasis remains challenging. We performed comparative proteomic and pathway analysis of four human metastatic HCC cell lines to identify metastasis-associated proteins. These HCC cell lines had a similar genetic background but with an increasing potential of metastasis. Using a combination of two dimensional electrophoresis (2-DE) and MALDI-TOF mass spectrometry, a total of 125 proteins and their post-translational modification forms or isoforms were found to be differentially expressed in the cell lines. Among them, 29 were gradually up-regulated whereas 17 were down-regulated with increasing metastatic potential. Instead of a traditional single-gene readout, global bioinformatics analysis was carried out, which revealed that the glycolysis pathway was the most significantly enriched pathway. The heat shock proteins (HSPs) centered and NF-kappaB centered networks were also enriched in the result, which may imply the key function of inflaming on metastasis. Meanwhile, knockdown of HDGF, an up-regulated protein and a target of NF-kappaB, induced cell apoptosis in the metastatic HCC cells. This work provides a demonstration that a combination of bioinformatics and comparative proteomics can help in finding out potential biomarkers associated with HCC metastasis on the level of pathways.

Human hepatocellular carcinoma (HCC) is one of the most malignant tumors, being particularly induced by unregulated growth and metastasis, and is a leading cause of death and major health problems in many countries. We report here the identification of 167 differentially expressed proteins between HCC (MHCC97-H) cells and Chang liver cells using enhanced nano-liquid chromatography/mass spectrometry (LC/MS). The most relevant pathways of differentially expressed proteins are involved in cytoskeleton organization, stress defense, and energy homeostasis etc. Moreover, of the identified proteins, there are 59 known or putative membrane-associated proteins with multitransmembrane domains confirmed by bioinformatic analysis. These proteins may be associated with cancer, reflecting tumorigenesis of HCC, and would be useful for the development of diagnostic and subsequently pharmaceutical targets of HCC. In addition, we identify a total of 41 proteins that are found to be up- or down-regulated following tanshinone IIA treatment for MHCC97H cells in a time-depended manner. Also, several proteins that are involved in actin cytoskeleton and stress resistance are mainly down-regulated, whereas proteins associated with cell redox homeostasis, mitochondrial, and microtubule-based movement are identified as mostly up-regulated after the treatment. Determination of functional roles of those differentially expressed proteins will enable further understanding of the mechanism of HCC tumorigenesis and exploration of new drugs for therapeutic intervention.

The extreme complexity of protein samples is becoming a great challenge for proteomic analysis, especially for those having large dynamic range of protein abundance. To solve this problem, and to overcome the limitation of the current proteomic technologies, a new method using hydrazide-functionalized magnetic microspheres was established in this study. With this method, tryptophan (Trp)-containing peptides can be selectively and sensitively enriched from complex and low-volume samples. Furthermore, combined with 1D-LC–MS/MS analysis, the strategy was successfully applied to the proteomic study of mouse serum. The proportion of Trp-containing peptides was increased from 19.4% to 80.2% through enrichment, and the complexity of the sample was reduced more than two times. An additional 113 Trp-containing peptides and 48 novel proteins were detected compared to the conventional method. This enrichment method provides a means for identifying more proteins as potential biomarkers in serum and other complex samples.

Endoglycosidase is a class of glycosidases that specifically cleaves the glycosidic bond between two proximal residues of GlcNAc in the pentasaccharide core of N-glycan, leaving the innermost GlcNAc still attached to its parent protein, which provides a different diagnostic maker for N-glycosylation site assignment. This study aims to validate the use of endoglycosidase for high throughput N-glycosylation analysis. An endoglycosidase of Endo H and the conventional PNGase F were employed, with a similar accessible procedure, for large-scale assignment of N-glycosylation sites and then N-glycoproteome for rat liver tissue. ConA affinity chromatography was used to enrich selectively high-mannose and hybrid glycopeptides before enzymatic deglycosylation. As a result, a total of 1063 unique N-glycosites were identified by nano liquid chromatography tandem mass spectrometry, of which 53.0% were unknown in the Swiss-Prot database and 47.1% could be assigned only by either of the methods, confirmed the possibility of large-scale glycoproteomics by use of endoglycosidase. In addition, 11 glycosites were assigned with core-fucosylation by Endo H. A comparison between the two enzymatic deglycosylation methods was also investigated. Briefly, Endo H provides a more confident assignment but a smaller dataset compared with PNGase F, showing the complementary nature of the two N-glycosite assignment methods.

A novel two-step protease digestion and glycopeptide capture approach has been developed. It is different from traditional tryptic digestion, glycopeptide enriching and identification approach in glycoproteomics. Here, proteins were first digested by Lys-C into relatively large peptides. Glycopeptides among them were selectively captured by hydrazide resin through oxidized glycans. After thorough washing steps, trypsin was used as a second protease to in situ release non-glycosylated part (named as LT-peptides) from glycopeptides. Subsequently, the remaining part of glycopeptides on resin was de-glycosylated by peptide-N-glycosidase F, and collected as DG-peptides. Finally, both LT- and DG-peptides could be analyzed by mass spectrometer, achieving glycoprotein and glycosite identification. The approach was applied to cell lysate after positive validation by a model glycoprotein: 143 N-glycoproteins identified from DG- and LT-fraction both. In those glycoproteins, 189 DG-peptide-revealed N-glycosites got further confirmation by neighboring LT-peptides, which, in the meantime, made 109 glycoproteins get improved sequence coverage with increase even up to 350% (averagely 79.4%). Through controllable release, separate identification and combined interpretation of non-glycopeptides (newly introduced LT-peptides here) and traditional de-glycopeptides, the approach could not only achieve routine N-glycosite identification, but also provide further proofs of N-glycosites and increase glycoprotein sequence coverage.

Secreted proteins are important sources for early detection and diagnosis of disease, and as such have received considerable attention. The extraction of low concentration proteins from large volumes of culture media, which are rich in salts and other compounds that interfere with most proteomics techniques, presents a problem for secretome studies. Ultrafiltration, precipitation, and dialysis are three major extraction methods that can be used to overcome this problem. The present study for the first time, compared the merits and shortcomings of these three methods, without bias. Centrifugal ultrafiltration provided the best extraction efficiency, and precipitation provided the highest number of identifiable proteins. The three methods yielded closely related, but different, information on the secretome; thus, they should be considered complementary or, at least, supplementary methods. Three hundred and sixty unique proteins were identified, including 211 potential secreted proteins. Compared with previous studies, this study also identified 42 new secreted proteins. The present study not only offers a reference for the selection of secretome extraction methods, but also expands the secretome database for the investigation of hepatocellular carcinoma.

A microfluidic reactor has been developed for rapid enhancement of protein digestion by constructing an alumina network within a poly(ethylene terephthalate) (PET) microchannel. Trypsin is stably immobilized in a sol–gel network on the PET channel surface after pretreatment, which produces a protein-resistant interface to reduce memory effects, as characterized by X-ray fluorescence spectrometry and electroosmotic flow. The gel-derived network within a microchannel provides a large surface-to-volume ratio stationary phase for highly efficient proteolysis of proteins existing both at a low level and in complex extracts. The maximum reaction rate of the encapsulated trypsin reactor, measured by kinetic analysis, is much faster than in bulk solution. Due to the microscopic confinement effect, high levels of enzyme entrapment and the biocompatible microenvironment provided by the alumina gel network, the low-level proteins can be efficiently digested using such a microreactor within a very short residence time of a few seconds. The on-chip microreactor is further applied to the identification of a mixture of proteins extracted from normal mouse liver cytoplasm sample via integration with 2D-LC-ESI-MS/MS to show its potential application for large-scale protein identification.

In this paper, a microchip-based sandwich-type aptasensor is developed for the detection of human thrombin. The SH-aptamer/thrombin/alkaline phosphatase-functionalized aptamer (ALP-aptamer) system was constructed in the microfluidic channels. And the substrate solution containing 4-aminophenyl phosphate (p-APP) was introduced to the microchannels for the end-column electrochemical detection. The on-chip aptasensor has a broad linear response range of 1–100 pM with a detection limit of 1 pM, which shows high sensitivity and specificity. The system was then applied to detect thrombin in human serum sample. Therefore, the on-chip aptasensor has a great promise for detecting and screening ultratrace levels of biomarkers in the complex matrices.

Herein, we report proteome and transcriptome profiles of the human adult liver and present an initial analysis. Overall, the human liver proteome (HLP) data set comprises 6788 identified proteins with at least two peptides matches at 95% confidence, including 3721 proteins newly identified in liver. The human liver transcriptome (HLT) data set consists of 11 205 expressed genes. The HLP is the largest proteome data set for a human organ and is the first direct association between a proteome and its transcriptome derived from the same sample. Although it is hard to approach complete coverage of the HLP currently, several conclusions based on this data set are clearly reached: (1) The 5816 protein-encoding genes (PEGs) represented by the HLP and the 11 104 PEGs represented in the HLT have been identified from 20 070 PEGs in IPI Human v3.07 and 19 478 PEGs in the integrated human transcriptome database, respectively. (2) The patterns of chromosomal distribution of the genes corresponding to the HLP are highly consistent with those of the HLT. Some chromosomal regions, such as 16p13.3, 19q13.31, 19q13.42, and Xq28, exhibit particularly high densities of liver-specific genes, which perform the important functions related to normal physiology or/and pathology in this organ. (3) The HLP spans 6 orders of magnitude in relative protein abundance and 78% of the proteins fall in the middle of this range. Of newly identified liver proteins, 82.5% are of low abundance. (4) Proteins involving in metabolism, transport, and coagulation and those containing active domains for metabolism, transport, and biosynthesis are significantly enriched in liver. (5) All 94 metabolic pathways in KEGG are touched to different extent. Of which, for 48 pathways, particularly those involved in metabolism of carbohydrates and amino acids, more than 80% of the component proteins have been detected. The liver-specific pathways, such as those participating in metabolism of bile acid and bilirubin and in biotransformation, are identified with remarkably high coverage. A total of 31 members of the cytochrome P450 family are identified, four of which have been observed for the first time in human liver. (6) Transport proteins involved in energy metabolism and secretion of both protein and bile acid are highly abundant. Three ion channels are described for the first time in liver. (7) The 800 proteins related to signal transduction and primarily involved in cellular recognition, localization, communication, and inflammation are present in the HLP data set. Insulin and adipocytokine pathways, which are involved in the regulation of glucose and fatty acids, are highly covered. (8) Transcription factors (309 in total) have been recognized at relatively low detection rates and abundance; however, transcription factors regulating gene expression related to transport, metabolism, and biosynthesis are detected at relatively higher coverage and the protein products of their target genes (100 in total), such as metabolic enzymes and plasma proteins, are also identified. (9) The overlap between the human liver and plasma proteomes is particularly noteworthy in the coagulation/anticoagulation/fibrinolysis and complement system. There is a significantly positive linear correlation between the abundance of coagulator proteins in liver and plasma.

A novel one-pipeline approach is reported, which can demonstrate glycoprotein identification and obtain intact glycosylation information after glycopeptide-level enrichment, without de-glycosylation. The proposed workflow has two enrichment steps plus two proteolytic processes: enriched glycoproteins were digested to peptides by Lys-C, and then enriched again and secondly digested by trypsin. In the resulting mixture, with a reasonable complexity, intact glycopeptides could be preserved and utilized informatively for glycosylation analysis, and non-glycopeptides for protein identification. In both standard protein mixture tests and real sample analysis, the resulting glycopeptides and non-glycopeptides were proved to play their expected roles, thus more confident protein glycosylation information was obtained.

A high-concentration with low viscosity ceramic silicon carbide (SiC) slurry prepared for ICP-OES determination of ultra-trace impurities is described in this paper. Good fluidity can be kept up to the slurry concentration as high as 30% (m v−1) by adding 2% polyethylene imine (PEI) as a dispersant at pH 4.0. Stability of the high content slurry of nm-size SiC is characterized by zeta potential measurement and viscosity measurement, and medium pH is experimentally optimized. The analysis can be calibrated using simple aqueous standards in case a high-concentration slurry is nebulized. Owing to extremely low blanks and unusually highly slurry concentration (20% m v−1), extraordinarily low limits of detection ranging from 2 (Mn, Ti) to 100 (Al) ng g−1 could be achieved.

In proteome research, rapid and effective separation strategies are essential for successful protein identification due to the broad dynamic range of proteins in biological samples. Some important proteins are often expressed in ultra low abundance, thus making the pre-concentration procedure before mass spectrometric analysis prerequisite. The main purpose of enrichment is to isolate target molecules from complex mixtures to reduce sample complexity and facilitate the subsequent analyzing steps. The introduction of nanoparticles into this field has accelerated the development of enrichment methods. In this review, we mainly focus on recent developments of using different nanomaterials for pre-concentration of low-abundance peptides/ proteins, including those containing post-translational modifications, such as phosphorylation and glycosylation, prior to mass spectrometric analysis.

Immobilization of enzyme on detonation nanodiamond (dND, 3–10 nm) and its application for efficient proteolysis have been demonstrated. By evaluation of the Michaelis constant (Km) and maximum velocity (Vmax) of immobilized enzyme, its activity was not impaired significantly by immobilization. And enzyme immobilized on dNDs exhibited much better thermal and chemical stabilities than its free counterpart and maintained high activity even after 10 times reuse. The efficient proteolysis by trypsin immobilized on dNDs (dND-trypsin) is demonstrated with the digestion of myoglobin (or other model protein) in a short time (5 min). Large numbers of identified peptides obtained by dNDs-trypsin enable a higher degree of sequence coverage and more positive identification of proteins than those obtained by in-solution digestion and the commercial immobilized trypsin beads, respectively. Moreover, immobilization of peptide-N-glycosidase F (PNGase F) on dNDs was realized and resulted in faster sequential glycosidase digestion of glycopeptides in less than 10 min.

This review gives a broad glance on the progress of recent advances on proteolysis and peptide/protein separation by chromatographic strategies in the past ten years, covering the main research in these areas especially in China. The reviewed research focused on enzymatic micro-reactors and peptide separation in bottom-up approaches, and protein and peptide separation in top-down approaches. The new enzymatic micro-reactor is able to accelerate proteolytic reaction rate from conventionally a couple of hours to a few seconds, and the multiple dimensional chromatographic-separation with various models or arrays could sufficiently separate the proteomic mixture. These advances have significantly promoted the research of protein/peptide separation and identification in proteomics.

A simple and sensitive method has been proposed to determine a trace level of α-fetoprotein (AFP), hepatocellular carcinoma biomarker, using poly(methyl methacrylate) (PMMA) microfluidic chips coupled with electrochemical detection system. The PMMA microchannels have been modified with poly(ethyleneimine) (PEI) containing abundant NH2 groups to covalently immobilize AFP monoclonal antibody. Afterward, the antigen AFP and horseradish peroxidase (HRP)-conjugated AFP antibody can sequentially bind through antigen–antibody specific interaction. Atomic force microscopy (AFM) and confocal fluorescence microscope (CFFM) were utilized to characterize the surface topography and protein immobilization after modification. Coupled with three-electrode electrochemical detection system, the immunochip can perform the detection limit of AFP down to 1 pg mL−1, and achieve a detectable linear concentration range of 1–500 pg mL−1 by differential pulse voltammetry (DPV). The on-chip immunoassay platform can not only provide rapid and sensitive detection for target proteins but also be resistant to non-specific adsorption of proteins, which contributes to the detection of low-level protein in real sample. Finally, AFP existing in healthy human serum was detected to demonstrate the utility of the immunochip. The result shows that the proposed approach is feasible and has the potential application in clinical analysis and diagnosis.

Gel-based matrix-assisted laser desorption ionization-time of flight tandem mass spectrometer (MALDI TOF/TOF MS) is one of the dominant methods of current proteomics, utilizing both peptide mass fingerprinting (PMF) and peptide fragment fingerprinting (PFF) for protein identification on a spot-to-spot basis. However, the unique impact of the quality of the corresponding mass spectrometry spectra remains largely unreported, and has motivated the development and use of an automatic spectra-assessment method. In this study, a multi-variant regression approach has been utilized to assess spectral quality for both PMF and PFF spectra obtained from MALDI TOF/TOF MS. The assessment index has been applied to investigations of MASCOT search results. Systematic examination of two large-scale sets of human liver tissue data has proved that spectral quality was a key factor in significant matching. Based on large-scale investigations on individual PMF search, individual PFF search and their combination, respectively, the filtering of bad quality spectra or spots proves to be an efficient way to improve search efficiency of all search modes in MASCOT. Meanwhile, a validation method based on score differences between normal and decoy (reverse or random) database searches is proposed to precisely define the positive matches. Further analysis showed that spectral quality assessment was also efficient in representing the quality of 2-DE gel spots and promoted the discovery of potential post-translation modifications.

Alternating current (AC) has been employed to enhance the efficiency of chymotryptic proteolysis for peptide mapping. It was allowed to flow through the mixture solution of proteins and chymotrypsin via a pair of platinum wire electrodes. Bovine serum albumin (BSA) and cytochrome c (Cyt-c) were digested by the novel proteolysis approach to demonstrate its feasibility and performance. The results indicated that AC significantly accelerated in-solution chymotryptic proteolysis and the digestion time was substantially reduced to 5 min. The digests were identified by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) with sequence coverages of 46% (BSA) and 90% (Cyt-c) that were much better than those obtained by using 12-h conventional in-solution chymotryptic proteolysis. In addition, AC-assisted chymotryptic proteolysis was employed to digest human serum to demonstrate its suitability to complex protein sample. The present proteolysis strategy is simple and efficient and will find a wide range of applications in proteomic research.

N-linked glycosylation is prevalent in proteins destined for extracellular environments; nearly all secreted proteins are glycosylated. However, with respect to their glycosylation sites, little attention has been paid. Here, we report the analysis of N-glycosylation sites on secreted proteins of human hepatocellular carcinoma cells. For the enrichment of glycopeptides, capture methods with hydrophilic affinity (HA) and hydrazide chemistry (HC) were used complementarily. With the use of both methods in combination with nano-LC-ESI-MS/MS analysis, 300 different glycosylation sites within 194 unique glycoproteins were identified, and 172 glycosites have not been determined experimentally previously. A direct comparison between HA and HC methods was also investigated for the first time. In brief, in terms of selectivity for glycopeptides, HC is superior to HA (92.9% vs 51.3%); however, based on the number of glycosites identified, HA outweighs HC (265 vs 159). Furthermore, unavoidable contaminants such as actin and bovine serum albumin which are not N-glycosylated could be easily depleted by using this glycoproteomic strategy. As a consequence, more low-abundance and genuinely secreted proteins were identified. Among the glycoproteins identified, α-fetoprotein, CD44 and laminin have been reported to be implicated in HCC and its metastasis.

A simple, rapid and reliable method for the determination of Al, Ca, Cr, Cu, Fe, Mg, Mn, Na, Ni, Si, Ti, V and Zr in high purity boron carbide (B4C) using slurry introduction axial viewed inductively coupled plasma optical emission spectrometry (ICP-OES) is described. The sample slurry was prepared by directly dispersing the powdery boron carbide in an aqueous solution without dispersant addition. The accuracy was verified by comparison of the results with those obtained by various other methods and the value of certified reference material ERM-ED 102 (boron carbide powder). Owing to a rather low contamination in the sample preparation and stability of the slurry, the LODs, which were in the range of 0.02–2 µg g−1, were superior to those of the conventional nebulization ICP-OES technique or solid dc-OES.

Detonation nanodiamond (dND) was firstly employed as adsorbent for pretreatment of peptides in dilute/contaminated sample solution. Detonation nanodiamond showed high efficiency for isolating and enriching peptides in a wide pH range. Remarkably, good tolerance capability toward salts and detergents could be achieved by using dNDs. Due to the inherent specificities of dNDs, dND-bound peptides could be directly analyzed by MALDI-TOF MS, so as to avoid the elution step and reduce sample loss. This pretreatment method also exhibited a better performance for protein identification compared to solvent evaporation and Ziptip pretreatment approach.

Glycosylation plays a key role in controlling various cellular processes; in diseases modifications of the glycans also highlight its clinical importance. However, ^glycosylation analysis remains a difficult task. In recent years, ^advances in sample preparation and mass spectrometry have greatly facilitated the analysis of glycoproteins. This review mainly covers five aspects of the improvements and advances on the research of protein glycosylation in China: 1) identification of glycoproteins, ^2) identification of glycosylation sites, ^3) new methods developed for glycopeptides enrichment, ^4) characterization of glycans, ^and 5) functional studies of protein glycosylation.

To investigate the differential proteins and related molecular mechanism of CDA-II (cell differentiation agent-II) induced therapy on a human hepatocellular carcinoma model in nude mice with high metastatic potential (LCI-D20).After tumors were transplanted 11 days, mice were intraperitoneally injected with CDA-II (1,800 mg/kg) for 20 days continuously. The tumor growth-inhibitory efficiency in CDA-II treated groups was calculated. Proteins extracted from tumor tissue were separated by two-dimensional gel electrophoresis (2DE) and the differential proteins were identified by matrix assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF-MS). Western blotting (WB) was performed to verify the expression of certain candidate proteins. Reverse transcription-polymerase chain reaction (RT-PCR) was engaged to study the molecular mechanism of the therapy.CDA-II suppressed the growth and metastasis of tumor. The tumor growth-inhibitory efficiency was 41.8%. In total, 27 differentially expressed proteins were identified, including HSP27, UGDH, CK8, Hsp60, ENOA and AnxA5, with functions involved in oncogene expression and/or cell differentiation. In addition, apparent alternations of HSP60 and β-actin expression levels and their different posttranslational modifications (PTMs) were investigated. RT-PCR analysis confirmed that the cancer related genes c-myc, N-ras and MMP-9 were significantly down-regulated.Our results demonstrate that CDA-II presence can change the proteome profiling and favors of the tumor suppression in LCI-D20 cell differentiation. Our results also suggest that the dynamic PTM of HSP60 expression levels could be used to predict HCC and might be a promising and useful biomarker to prognosticate CDA-II therapeutic efficacy.

In this work, aminophenylboronic acid-functionalized magnetic nanoparticles were synthesized, and applied to selective separation of glycopeptides and glycoproteins.

Preeclampsia (PE) is a complex and serious condition of pregnancy. Trophoblasts in human placenta can be separated and collected by laser capture micro-dissection (LCM). Protein in trophoblasts have been extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), finally 962 unique proteins are identified by liquid chromatography coupled to mass spectrometry (LC–MS/MS). Comparison of differential expressed proteins in normal and those in PE are investigated. Two-dimensional electrophoresis (2DE) and MS were used to identify differential expressed proteins. 13 differential expressed proteins include signal transduction protein, molecular chaperone, cell skeleton proteins are identified, in which 3 proteins are down-regulated and 10 proteins are up-regulated. They might be correlated with the cause of PE.

Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is a standard analytical tool for protein identification and peptide sequencing. High sensitivity and resolution are two critical parameters for recording good peptide mass fingerprinting (PMF) of low abundance proteins. Here, we report a novel nanodiamond (ND) (normal size 3–10 nm) support for MALDI-MS target, over which α-cyano-4-hydrocinnamic acid (CCA) crystallizes evenly. Good reproducibility of relative peak intensity (R.S.D. less than 11.8%) among sample spot (from ring to center) is achieved on ND support. Therefore, the search for “hot spots” during the analysis is not necessary, which is supporting for the automatic acquisition of data. Due to high absorbability of energy from the laser, the ND support improves ionization efficiency of samples. In general, the sensitivity of MS obtained on ND support can be enhanced three to four times compared to the conventional MALDI sample preparation technique. Sensitivity obtained on ND support ranges from 62.5 amol of Arg-vasopressin standard peptide to 1.0 fmol of myoglobin tryptic peptide mixture. Reduced spot size and increased sensitivity in MALDI-MS are also accomplished by ND support. With spot size reduced, the signal intensity of cytochrome c (Cyt c) tryptic peptide obtained on ND support is at least seven times greater than it acquired on stainless steel. And ND support has been found better tolerance for salt (up to 500 mM NaCl) to MALDI-MS analysis. All these properties make ND support a valuable tool for MALDI-MS identification of proteins.

A novel technique of simultaneous peptide enrichment and wash-free in-situ self-desalting for MALDI analysis is reported, where a newly synthesized block copolymer with a microphase-separated configuration is applied to embed salts with its hydrophilic domain of poly(ethylene oxide) and concentrate peptides with its hydrophobic domain of polysulfone.

An enzymatic microreactor has been fabricated based on the poly(methyl methacrylate) (PMMA) microchchip surface-modified with zeolite nanoparticles. By introducing the silanol functional groups, the surface of PMMA microchannel has been successfully modified with silicalite-1 nanoparticle for the first time due to its large external surface area and high dispersibility in solutions. Trypsin can be stably immobilized in the microchannel to form a bioreactor using silica sol–gel matrix. The immobilization of enzyme can be realized with a stable gel network through a silicon–oxygen–silicon bridge via tethering to those silanol groups, which has been investigated by scanning electron microscopy and microchip capillary electrophoresis with laser-induced fluorescence detection. The maximum proteolytic rate constant of the immobilized trypsin is measured to be about 6.6 mM s−1. Using matrix assisted laser desorption and ionization time-of-flight mass spectrometry, the proposed microreactor provides an efficient digestion of cytochrome c and bovine serum albumin at a fast flow rate of 4.0 µL min−1, which affords a very short reaction time of less than 5 s.

A novel method for the determination of Al, Ca, Cr, Cu, Fe, Mg, Mn, Ni and Ti in high purity silicon carbide (SiC) using slurry introduction axial viewed inductively coupled plasma optical emission spectrometry (ICP-OES) was described. The various sizes of SiC slurry were dispersed by adding dispersant polyethylene imine (PEI). The stability of slurry was characterized by zeta potential measurement, SEM observation and signal stability testing. The optimal concentration of PEI was found to be 0.5 wt% for the SiC slurry. Analytical results of sub-μm size SiC by the slurry introduction were in good accordance with those by the alkaline fusion method which verified that determination could be calibrated by aqueous standards. For μm size SiC, results of most elements have a negative deviation and should be calibrated by the Certified Reference Material slurry. Owing to a rather low contamination in the sample preparation and stability of the slurry, the limits of detection (LODs), which are in the range of 40–2000 ng g−1, superior to those of the conventional nebulization technique by ICP-OES or ICP-MS.

Sample preparation is still the first and important step toward successful two-dimensional gel electrophoresis (2DE) and identification in proteomics study. The 2DE profiling of eggs of silkworm specie by using conventional one-step extraction, however, is unsatisfactory because high-abundance proteins such as egg-specific protein (ESP) and No 30 family (30 KP) in the extract lead to difficulties in detecting most of biologically relevant proteins. Based on the tendency of these abundant proteins to be soluble in Tris–HCl buffer, we report herein a robust approach in which the extract enriched in ESP and 30 KP was fractionationed and mixed with the re-extract of residual pellet in an optimal proportion. In comparison with the one-step method, the 2DE pattern was improved by this new method with over one-third enhancement in spots. A total of 48 unique proteins obtained have been furthermore identified by mass spectrometry (MS) and MS/MS. The identified proteins are found to include heat shock proteins families, ribosomal proteins, disulfide isomerase proteins, Glutathione S-transferase, and elongation factor, etc., which are mainly involved in some important processes. To our knowledge, this is the first time that the several proteins have been detected in silkworm eggs by proteomics means. This simple and reproducible approach would raise the opportunity of discovering and identifying more biomarkers and determining their possible roles in further studies.

Characterization of the stability of ceramic suspension for slurry introduction in inductively coupled plasma optical emission spectrometry (ICP-OES) and its application to the analysis of aluminium nitride (AlN) was reported in this paper. Stability and homogeneity suspensions of μm sized AlN and nm sized AlN, as the representatives selected, were characterized by sedimentation test, scanning electron microscope observation and zeta potential measurement. The zeta potential of the interface between the particle and the medium can be controlled by pH adjustment and adding 0.8 wt% polyethylene imine (PEI) for μm size AlN or 2.0 wt% PEI for nm size AlN, respectively. The analytical results obtained via slurry introduction were compared with those via nebulization of aqueous solutions prepared by fusion digestion. The nm size AlN can be directly analyzed by using slurry nebulization ICP-OES with aqueous standards calibration, and the impurities Cu, Fe, Mg, Mn, etc. in μm size AlN can also be determined by slurry method, but the results of some elements with high boiling points, such as Si, Y, etc., show deviation from the reference values.

A method of slurry nebulization for inductively coupled plasma optical emission spectrometry (ICP-OES) applied to the analysis of titanium niride(TiN) was reported. The TiN slurry sample was prepared with adding dispersant polyacrylate amine or polyethylene imine for the stabilization and homogenization of suspension, and the amount of additives was optimized. A Babington type cross-flow nebulizer with V-groove was used for nebulization of the slurry for avoidance of blockage from the particles. The stability of slurry was characterized via zeta potential measurement, scanning electron microscope observation, particle size distrbution measurement and signal stability testing. For nm size TiN, calibration curves could be established by aqueous standards and the analytical results were in good accordance with the alkaline fusion method. For μm size TiN, a negative deviation was observed for most of elements and this deviation can be corrected by using Ti intrinsic internal standard method.

Metal-ion-immobilized zeolite nanoparticles have been applied for the first time to isolate phosphopeptides from tryptic β-casein digest; the phosphopeptides enriched on the modified zeolite nanoparticles could be effectively identified by MALDI-TOF-MS/MS.

The design and characterization of two kinds of poly(dimethylsiloxane) (PDMS) microfluidic enzymatic-reactors along with their analytical utility coupled to MALDI TOF and ESI MS were reported. Microfluidic devices integrated with microchannel and stainless steel tubing (SST) was fabricated using a PDMS casting technique, and was used for the preparation of the enzymatic-reactor. The chemical modification was performed by introducing carboxyl groups to PDMS surface based on ultraviolet graft polymerization of acrylic acid. The covalent and physical immobilization of trypsin was carried out with the use of the activation reagents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) and a coupling reagent poly(diallyldimethylammonium chloride) (PDDA), respectively. The properties and success of processes of trypsin immobilization were investigated by measuring contact angle, infrared absorption by attenuated total reflection spectra, AFM imaging and electropherograms. An innovative feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by embedded SST electrode and replaceable tip. The lab-made devices provide an excellent extent of digestion of several model proteins even at the fast flow rate of 3.5 µL min−1 for the EDC/NHS-made device and 0.8 µL min−1 for the PDDA-made device, which afford very short residence times of 5 s and 20 s, respectively. In addition, the lab-made devices are less susceptive to memory effect and can be used for at least 50 runs in one week without noticeable loss of activity. Moreover, the degraded PDDA-made device can be regenerated by simple treatment of a HCl solution. These features are the most required for microfluidic devices used for protein analysis.

A slurry sample introduction technique for the analysis of TiO2 powders by axial viewing ICP-OES is reported. Relationships between stable slurries and the conditions, such as particle size, pH, dispersant and its amount, were experimentally investigated. Slurries were prepared by mixing the TiO2 powder with the dispersant poly(acrylate amine) (NH4PAA) and agitation in an ultrasonic bath to ensure good dispersion. The pH of the medium and amount of dispersant has been optimized. Impurities in TiO2 were determined by an axial viewing ICP-OES spectrometer. As representatives, a Standard Reference Material 154 b of µm particle size TiO2 and a nm particle size TiO2 were selected and determined for micro and trace impurities. Analytical results obtained via slurry sample introduction were compared with those via nebulization of aqueous solutions prepared by acid digestion and fusion digestion. The determined results by slurry introduction were in good accordance with the stated values, which verified that the calibration curves could be established by aqueous standards.

Functionalized large-pore mesoporous SBA-15 is utilized for the first time as a good substrate in high performance liquid chromatography (HPLC) to separate biomolecules including peptides and proteins.

This paper reports a quantitative electronic nose (enose) for the quantitative determination of Freon gas within the concentration range 0–1000 ppm in the presence of interfering gases such as water, lubricant and petrol vapours. This quantitative enose is a new type of Freon detection system, composed of an array of four sensors. The artificial neural network (ANN) and fuzzy logic type of ANN (FNN), in combination with the relative error concept in analytical chemistry, are integrated for both quantification and discrimination. The predicted results are satisfied with a pass rate of >80% within the permitted relative errors. The results show that the Freon enose developed in this study is reliable for both the qualitative and quantitative determination of Freon gas and exhibits the merits of high sensitivity, anti-interference and accuracy.

The fabrication and application of an end-column amperometric detection (AD) system with a carbon electrode for capillary-column liquid chromatography (CLC) were described. This new amperometric detector showed good sensitivity and stability in detecting the thiocompounds and other analytes with CLC. In order to obtain the better separation and detection performance for analytes, several operational parameters had been investigated: the working potentials, pH and flow rate. Under the optimum conditions, the method could effectively separate and determine cysteine (Cys), glutathione (GSH), dopamine (DA) and 6-thiopurine (6-TP). Good repeatability for retention time was obtained with a relative standard deviation (R.S.D.) value of 0.5%. The linear range covered over three orders of magnitude and the limits of detection were 8 fmol for cysteine, 20 fmol for glutathione, 8 fmol for dopamine and 20 fmol for 6-thiopurine. This method was successfully applied in determination of urinary sample with characteristics of simplicity, high sensitivity and good repeatability.

•Increased number of identified proteins compared to the 12 h in-solution digestion.•Enhanced proteolytic performance towards a group of proteins with specific pI.•Desirable throughput consuming ∼6 min for proteolysis of bio-samples.•Automated process in the sample pretreatment for online MS analysis.•Long term stability after two months storage, reusability with little memory effect.Mass spectrometry (MS)-based proteome profiling is essential for molecular diagnostics in modern biomedical study. To date, sample preparation including protein extraction and proteolysis is still very challenging and lack of efficiency. Recently tips-based sample preparation protocols exhibit strong potentials to achieve the goal of “a proteome in an hour”. However, in-tip proteolysis is still rarely reported and far from ideal for dealing with complex bio-samples. In this work, nanoreactors encapsulated micropipette tips were demonstrated as high performance devices for fast (∼minutes) and multiplexing proteolysis to assist the profiling of cancer cells proteome. Nanoporous silica materials with controlled pore size and surface chemistry were prepared as nanoreactors and encapsulated in micropipette tips for efficient in situ proteolysis. The as-constructed device showed desirable sensitivity (LOD of 0.204 ± 0.008 ng/μL and LOQ of 0.937 ± 0.055 ng/μL), selectivity, stability (two months under −20 °C), reusability (at least 10 times), and little memory effect in MS based bottom-up proteomic analysis. It was used for comprehensive protein mapping from cancer cell lines. The number of identified proteins was increased by 18%, 22%, 52%, and 52% dealing with HepG2, F56, MCF7, and HCCLM3 cancer cells, compared to traditional in-solution proteolysis based bottom-up proteomic strategy. With the enhanced performance, our work built a novel, efficient and miniaturized platform for facile proteomic sample preparation, which is promising for advanced biomarkers discovery in biomedical study.

In this work, aminophenylboronic acid-functionalized magnetic nanoparticles were synthesized, and applied to selective separation of glycopeptides and glycoproteins.

A novel technique of simultaneous peptide enrichment and wash-free in-situ self-desalting for MALDI analysis is reported, where a newly synthesized block copolymer with a microphase-separated configuration is applied to embed salts with its hydrophilic domain of poly(ethylene oxide) and concentrate peptides with its hydrophobic domain of polysulfone.

The dispersive effect of polyacrylate amine (NH4PAA) upon suspensions of silicon nitride (Si3N4) is considered. Slurry introduction for axial inductively coupled plasma optical emission spectrometry determination of Al, Ca, Co, Cr, Cu, Fe, K, Mg, Mn, Na, Ni and Ti in powdered Si3N4 has also been investigated. Characterization by zeta potential measurement and slurry stability measurements shows that NH4PAA is a good dispersant for the preparation of Si3N4 suspensions. The optimal concentration of NH4PAA was 0.8 wt% for μm-sized Si3N4 and 1.2 wt% for nm-sized Si3N4. The analytical results obtained using nm- and μm-sized Si3N4 by slurry introduction were in good accordance with results obtained by the high-pressure acid deposition method, thus verifying that calibration curves could be established using aqueous standards. By introducing a low amount of contamination in the sample preparation, an accurate result is obtained for the detection of contamination-risk elements such as K and Na, which cannot be accurately determined by the high-pressure acid deposition method. The limits of detection, which are in the range of 8–250 ng g−1, were shown to be superior to those of the conventional nebulization technique with ICP-OES or ICP-MS.

A simple, rapid and reliable method for the determination of Al, Ca, Cr, Cu, Fe, Mg, Mn, Na, Ni, Si, Ti, V and Zr in high purity boron carbide (B4C) using slurry introduction axial viewed inductively coupled plasma optical emission spectrometry (ICP-OES) is described. The sample slurry was prepared by directly dispersing the powdery boron carbide in an aqueous solution without dispersant addition. The accuracy was verified by comparison of the results with those obtained by various other methods and the value of certified reference material ERM-ED 102 (boron carbide powder). Owing to a rather low contamination in the sample preparation and stability of the slurry, the LODs, which were in the range of 0.02–2 µg g−1, were superior to those of the conventional nebulization ICP-OES technique or solid dc-OES.

A high-concentration with low viscosity ceramic silicon carbide (SiC) slurry prepared for ICP-OES determination of ultra-trace impurities is described in this paper. Good fluidity can be kept up to the slurry concentration as high as 30% (m v−1) by adding 2% polyethylene imine (PEI) as a dispersant at pH 4.0. Stability of the high content slurry of nm-size SiC is characterized by zeta potential measurement and viscosity measurement, and medium pH is experimentally optimized. The analysis can be calibrated using simple aqueous standards in case a high-concentration slurry is nebulized. Owing to extremely low blanks and unusually highly slurry concentration (20% m v−1), extraordinarily low limits of detection ranging from 2 (Mn, Ti) to 100 (Al) ng g−1 could be achieved.

Slurry nebulization in plasmas has advantages of simplicity, high speed, low cost, minimized analyte loss, and low risk of sample contamination, but it has not been very widely adopted. However, the study of advanced ceramic materials has recently renewed enthusiasm for this technique. In this paper, the current state of research on slurry nebulization in plasmas for the analysis of advanced materials is thoroughly surveyed. Sample preparation, sample characterization, and modifications of the instrumentation necessary for slurry nebulization are reviewed, along with calibration procedures and studies on fundamental issues. Finally, the applications of this method to a variety of advanced materials are summarized, and the outlook for this method is discussed.

A general and effective enzymatic labeling method, termed glycan reducing end dual isotopic labeling (GREDIL), was developed for mass spectrometry-based quantitative N-glycomics.

A segment flow protocol has been proposed for coupling nano-liquid chromatography (nano-LC) with mass spectrometry (MS) in peptide-based proteomics research. LC effluent was segmented by perfluorodecalin oil and stored as segments in sequence to carry out the following off-line MSn analysis. In this way, incomplete identification of co-eluted peptides resulting from the MS scan rate, relatively slower than the LC elution rate, was solved to some extent for on-line coupling LC-MSn analysis. The complex proteins from a sample of T. tengcongensis were successfully identified with one run using segment as the interface between nano-LC and MSn analysis, compared to the total identified proteins with six runs using routine on-line nano-LC-MSn analysis. This segment flow approach could eliminate detection loss and sample cross-contamination, providing a new method for peptide-based proteomic research, especially when the amount of sample is limited.

The liver is an important organ and is the biggest digestive gland in the human body. The establishment of a proteome database of the human liver would provide useful information for human liver disease treatment. In order to maximize protein identification and to compare different analyses, multiple technical routes were used for proteome profiling of French liver samples. Five strategies, including two two-dimensional electrophoresis (2DE) methods and three multi-dimensional liquid chromatography (MDLC) methods were evaluated. By using these five routes, 1627 unique proteins were finally identified. Various bioinformatics analyses focused on physicochemical properties and functional classification were used to examine the characteristics of the protein expression profile. Furthermore, comparison of these data with an existing liver expression profile provided by UNIGENE and UNIPROT allowed us to investigate the identification efficiency of this dataset. The different technical methods were evaluated and compared to make a model of the organ proteome profile.

Lectin microarrays are increasingly used as screening tools with a high potential for glycobiology analysis, providing a high-sensitivity profiling of complex glycan features in a short time, without the need for the release of glycan. The application of the technology for the rapid differential glycan profiling of target samples resulted in the discovery of new glycan-related biomarkers for disease detection and particular types of glycobiology studies. Additionally, lectin microarrays might offer many opportunities for further innovation, and their range of applications in the life sciences promises to be greatly expanded. Herein, we describe the advances in the fabrication and detection of lectin microarray technologies and their application, which herald even more exciting breakthroughs in the coming decade.