There are two challenges in oligonucleotide detection by liquid chromatography coupled with mass spectrometry (LC-MS), the serious ion suppression effects caused by ion-pair reagents and the low detection sensitivity in positive mode MS. In this study, highly concentrated alcohol vapors were introduced into an enclosed electrospray ionization chamber, and oligonucleotides could be well detected in negative mode MS even with 100 mM triethylammonium acetate (TEAA) as an ion-pair reagent. The MS signal intensity was improved 600-fold (for standard oligonucleotide dT15) by the isopropanol vapor assisted electrospray, and effective ion-pair LC separation was feasibly coupled with high-sensitive MS detection. Then, oligonucleotides were successfully detected in positive mode MS with few adducts by propanoic acid vapor assisted electrospray. The signal intensity was enhanced more than 10-fold on average compared with adding acids into the electrospray solution. Finally, oligonucleotides and peptides or histones were simultaneously detected in MS with little interference with each other. Our strategy provides a useful alternative for investigating the biological functions of oligonucleotides.

Homogeneous gold nanoclusters (Au NCs) have been widely utilized in drug delivery, chemical sensing, bioassays, and biolabeling due to their unique physical and chemical properties. However, little attention has been paid to their application in detecting protein post-translational modifications. Herein, we describe the development of a homogeneous reaction system with water-soluble zwitterionic Au NCs to capture glycopeptides from complex biological samples. The unique characteristics of Au NCs, such as their molecular-like properties, the excellent homogeneity in aqueous solution, the organic solvent responsive precipitation, and the easy preparation in only 4.5 h, contribute to the high efficiency and high throughput for capturing the targeted glycopeptides. Compared with the conventional heterogeneous system with solid-state adsorbents, the number of characterized glycosylation sites was improved by 35%. Finally, an MS detection limit as low as 50 amol was achieved for the standard glycoprotein (IgG), and 1576 glycosylation sites from 713 glycoproteins were identified from only 60 μg of mouse liver protein. Data are available via ProteomeXchange with identifier PXD005635.

•Electrospray ionization is performed in the presence of concentrated ACN vapor.•The background interferences are feasibly suppressed and the peptide signals are enhanced in ESI–MS analyses.•The median MS signal intensity was improved 5 times for the 23 BSA tryptic peptides.•The number of reproducibly and accurately quantified peptides could be improved 67% in six replicate label-free quantitative proteome analyses of cell line sample.Suppressing the background interferences and enhancing the analytes signals are long-term goals in high performance electrospray ionization mass spectrometry (ESI–MS) analyses. We observed that performing electrospray in the presence of a concentrated acetonitrile atmosphere suppresses background interferences and enhances peptide signals. An enclosed nanoESI source was utilized to provide a stable atmosphere of concentrated acetonitrile vapor for high performance ESI–MS analyses. The median MS signal intensity increased by 5 times for a set of 23 BSA tryptic peptides in direct ESI–MS analysis. Further, the number of reproducibly and precisely quantified peptides could be improved 67% in six replicate label-free quantitative proteome analyses by this strategy.

Homogeneous and water-soluble Au nanoclusters with high UV photon adsorption and high biocompatibility were utilized to enhance the detection sensitivity and lateral resolution of multiplexed mass spectrometry imaging based on laser desorption/ionization.

•A competitive substitution strategy was developed to enrich multi-phosphopeptides.•Different pre-coordinated phosphate salts have distinct selectivity shifts.•The surface charge of pre-coordinated adsorbents is important to the selectivity.•85% selectivity to multi-phosphopeptides was feasibly obtained for complex sample.•Sequential enrichment of mono- and multi-phosphopeptides was achieved.Although many affinity adsorbents have been developed for phosphopeptides enrichment, high-specifically capturing the multi-phosphopeptides is still a big challenge. Here, we investigated the mechanism of phosphate ion coordination and substitution on affinity adsorbents surfaces and modulated the selectivity of affinity adsorbents to multi-phosphopeptides based on the different capability of mono- and multi-phosphopeptides in competitively substituting the pre-coordinated phosphate ions at strong acidic condition. We demonstrated both the species of pre-coordinated phosphate ions and the substituting conditions played crucial roles in modulating the enrichment selectivity to multi-phosphopeptides, and the pre-coordinated affinity materials with relative more surfaces positive charges exhibited better enrichment efficiency due to the cooperative effect of electrostatic interaction and competitive substitution. Finally, an enrichment selectivity of 85% to multi-phosphopeptides was feasibly achieved with 66% improvement in identification numbers for complex protein sample extracted from HepG2 cells. Data are available via ProteomeXchange with identifier PXD004252.

Positively charged lysines are crucial to maintaining the native structures of proteins and protein complexes by forming hydrogen bonds and electrostatic interactions with their proximal amino acid residues. However, it is still a challenge to develop an efficient method for probing the active proximal microenvironments of lysines without changing their biochemical/physical properties. Herein, we developed an active covalent labeling strategy combined with mass spectrometry to systematically probe the lysine proximal microenvironments within membrane protein complexes (∼700 kDa) with high throughput. Our labeling strategy has the advantages of high labeling efficiency and stability, preservation of the active charge states, as well as biological activity of the labeled proteins. In total, 121 lysines with different labeling levels were obtained for the photosystem II complexes from cyanobacteria, red algae, and spinach and provided important insights for understanding the conserved and nonconserved local structures of PSII complexes among evolutionarily divergent species that perform photosynthesis.

Magnetic nanoparticles functionalized with maltosylated glycopeptide dendrimers were prepared via azide-alkynyl click reaction. The functionalized magnetic nanoparticles exhibited high hydrophilicity and good efficiency in glycopeptide enrichment by HILIC.

We develop an acidic vapor assisted electrospray ionization strategy within an enclosed electrospray ionization source to counteract the ion suppression effects caused by trifluoroacetic acid. The mass spectrometry signal intensity of intact proteins was improved 10 times and the number of valid signals for E. coli intact protein samples was improved 96% by using this strategy.

N-dodecyl β-D-maltoside (DDM), a mild detergent with the ability to maintain the enzyme activity and solubilize hydrophobic proteins without changing their structures, was applied for N-glycoproteomic analysis of minute protein sample from mouse brain tissue. After combining with the capillary-based glycoproteomic reactor, 281 N-glycosylation sites were successfully characterized from 50 μg of mouse brain tissue, which was 110% higher at least than those obtained by conventional strategies.

•A novel adsorbent coated with branched PEI and maltose was prepared.•The adsorbent exhibited high hydrophilicity and selectivity.•The adsorbent exhibited high N-glycopeptide enrichment capacity (>150 mg/g, IgG/MNPs).•The adsorbent exhibited high N-glycopeptide enrichment recovery (85%).•1567 N-glycopeptides were enriched from the complex protein sample.Hydrophilic interaction chromatography (HILIC) adsorbents have drawn increasing attention in recent years due to their high efficiency in N-glycopeptides enrichment. The hydrophilicity and binding capacity of HILIC adsorbents are crucial to the enrichment efficiency and mass spectrometry (MS) detection sensitivity of N-glycopeptides. Herein, magnetic nanoparticles coated with maltose-functionalized polyethyleneimine (Fe3O4-PEI-Maltose MNPs) were prepared by one-pot solvothermal reaction coupled with “click chemistry” and utilized for N-glycopeptides enrichment. Owing to the presence of hydrophilic and branched polyethyleneimine, the amount of immobilized disaccharide units was improved about four times. The N-glycopeptides capturing capacity was about 150 mg/g (IgG/MNPs) and the MS detection limitation as low as 0.5 fmol for IgG and 85% average enrichment recovery were feasibly achieved by using this hybrid magnetic adsorbent. Finally, 1237 unique N-glycosylation sites and 1567 unique N-glycopeptides from 684 N-glycoproteins were reliably characterized from 60 μg protein sample extracted from mouse liver. Therefore, this maltose-functionalized polyethyleneimine coated adsorbent can play a promising role in highly efficient N-glycopeptides enrichment for glycoproteomic analyses of complex protein samples.

Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.

Although glycoproteomics is greatly developed in recent years, our knowledge about N-glycoproteome of human tissues is still very limited. In this study, we comprehensively mapped the N-glycosylation sites of human liver by combining click maltose–hydrophilic interaction chromatography (HILIC) and the improved hydrazide chemistry. The specificity could be as high as 90% for hydrazide chemistry and 80% for HILIC. Altogether, we identified 14 480 N-glycopeptides matched with N-!P-[S|T|C] sequence motif from human liver, corresponding to 2210 N-glycoproteins and 4783 N-glycosylation sites. These N-glycoproteins are widely involved into different types of biological processes, such as hepatic stellate cell activation and acute phase response of human liver, which all highly associate with the progression of liver diseases. Moreover, the exact N-glycosylation sites of some key-regulating proteins within different human liver physiological processes were also obtained, such as E-cadherin, transforming growth factor beta receptor and 29 members of G protein coupled receptors family.

Hepatocellular carcinoma is one of the most fatal cancers worldwide. In this study, a reversed-phase–strong cation exchange–reversed-phase three-dimensional liquid chromatography strategy was established and coupled with mass spectrometry to investigate the differential proteome expression of HCC and normal liver tissues. In total, 2759 proteins were reliably quantified, of which, 648 proteins were dysregulated more than 3-fold in HCC liver tissues. Some important proteins that relate to HCC pathology were significantly dysregulated, such as NAT2 and AKR1B10. Furthermore, 2307 phosphorylation sites from 1264 phosphoproteins were obtained in our previous phosphoproteome quantification, and the nonphosphorylated counterparts of 445 phosphoproteins with 983 phosphorylation sites were reliably quantified in this work. It was observed that 337 (34%) phosphorylation sites exhibit significantly different expression trends from that of their corresponding nonphosphoproteins. Some novel phosphorylation sites with important biological functions in the progression of HCC were reliably quantified, such as the significant downregulation of pT185 for ERK2 and pY204 for ERK1.

Conventional N-glycoproteome analysis usually applies C18 reversed-phase (RP) adsorbent for sample purification, which will lead to unavoidable sample loss due to the high hydrophilicity of N-glycopeptides. In this study, a porous graphitized carbon (PGC) absorbent was combined with a C18 adsorbent for N-glycopeptide purification in comprehensive N-glycoproteome analysis based on the hydrophobic and polar interactions between carbon and N-glycans. It was observed that the small hydrophilic N-glycopeptides that cannot retain onto C18 adsorbent can be captured by the graphitized carbon, while the large hydrophobic N-glycopeptides that cannot retain onto the graphitized carbon can be feasibly captured by the C18 adsorbent. Comparing with sample purification by using C18 adsorbent only, 28.5 % more N-glycopeptides were identified by combining both C18 and PGC adsorbents. The C18-PGC strategy was further applied for both sample purification and pre-fractionation of a complex protein sample from HeLa cell. After hydrophilic interaction chromatography enrichment, 1,484 unique N-glycopeptides with 1,759 unique N-glycosylation sites were finally identified.

Despite the importance of protein N-glycosylation in a series of biological processes, in-depth characterization of protein glycosylation is still a challenge due to the high complexity of biological samples and the lacking of highly sensitive detection technologies. We developed a monolithic capillary column based glycoproteomic reactor enabling high-sensitive mapping of N-glycosylation sites from minute amounts of sample. Unlike the conventional proteomic reactors with only strong-cation exchange or hydrophilic-interaction chromatography columns, this novel glycoproteomic reactor was composed of an 8 cm long C12 hydrophobic monolithic capillary column for protein digestion and a 6 cm long organic–silica hybrid hydrophilic monolithic capillary column for glycopeptides enrichment and deglycosylation, which could complete whole-sample preparation including protein purification/desalting, tryptic digestion, enrichment, and deglycosylation of glycopeptides within about 3 h. The developed reactor exhibited high detection sensitivity in mapping of N-glycosylation sites by detection limit of horseradish peroxidase as low as 2.5 fmol. This reactor also demonstrated the ability in complex sample analysis, and in total, 486 unique N-glycosylation sites were reliably mapped in three replicate analyses of a protein sample extracted from ∼104 HeLa cells.

•The separation of intact proteins on the POSS-based hybrid monolithic capillary column was investigated.•The POSS-based monolithic capillary columns exhibits good permeability and can be operated at high flow rate with slight decrease of the column efficiency.•Fast separation of seven intact proteins can be realized in 2.5 min.High-efficient separation of intact proteins is still a huge challenge in proteome analysis of complex biological samples by using capillary columns. In this study, four POSS-based hybrid monolithic capillary columns were prepared and applied in nano-flow liquid chromatography (Nano-LC) separation of intact proteins. It was observed that the POSS-based hybrid monolithic columns exhibit high permeability, good LC separation reproducibility and column efficiency for intact protein separation. The effects of different LC separation conditions such as flow rate, gradient steepness, column length and mobile phase additives on the LC separation efficiency of the POSS-based hybrid monolithic column were systematically examined. Finally, fast LC separation of 7 proteins mixture was realized in 2.5 min by using the optimized conditions on the 100 μm i.d. POSS-based hybrid monolithic capillary column.

The titanium silicalite-1 (TS-1) with post-treatment of chemical selective desilication (T-TS-1) has been synthesized, characterized and applied as a potential adsorbent for selective capture of phosphopeptides from complex biological samples prior to mass spectrometry analysis. A T-TS-1 material-based successive phosphopeptide enrichment strategy has also been established for real biological sample analysis, which exhibited high enrichment selectivity and is comparable to existing materials.

We developed a reversed-phase monolithic column based enzyme reactor for protein analysis. The effects of buffer solution, porous structure of the column, digestion time and hydrophobicity of solid phase on the digestion performance of enzyme reactor were systematically investigated in terms of the number of identified unique peptides, sequence coverage and the level of missed cleavage as the evaluation indicators. The results demonstrated that the reversed-phase monolithic column based enzyme reactor had high digestion efficiency and might be further extended to the proteome analysis of complex samples.Base peak chromatogram for separation of 1 μg BSA digest by C12 monolithic column microreactor. This new enzyme reactor showed more base peaks and higher peak intensity in the peak chromatogram compared with C18 monolithic column and C18 packed column microreactors.

•All the proteins' relative abundances were adjusted into 2 orders of magnitude (1/9-9).•The quantification accuracy and coverage of extreme proteins and protein phosphorylation sites had been improved.•The newly expressed or disappeared proteins and protein phosphorylation sites could be accurately quantified.•The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times.Mass spectrometry (MS) based quantitative analyses of proteome and proteome post-translational modifications (PTMs) play more and more important roles in biological, pharmaceutical and clinical studies. However, it is still a big challenge to accurately quantify the proteins or proteins PTM sites with extreme relative abundances in comparative protein samples, such as the significantly dysregulated ones. Herein, a novel quantification strategy, Mixing at Specific Ratio (MaSR) before isotope labeling, had been developed to improve the quantification accuracy and coverage of extreme proteins and protein phosphorylation sites. Briefly, the comparative protein samples were firstly mixed together at specific ratios of 9:1 and 1:9 (w/w), followed with mass differentiate light and heavy isotope labeling, respectively. The extreme proteins and protein phosphorylation sites, even if the newly expressed or disappeared ones, could be accurately quantified due to all of the proteins' relative abundances had been adjusted to 2 orders of magnitude (1/9-9) by this strategy. The number of quantified phosphorylation sites with more than 20 folds changes was improved about 10 times in comparative quantification of pervanadate stimulated phosphoproteome of HeLa cells, and 134 newly generated and 21 disappeared phosphorylation sites were solely quantified by the MaSR strategy. The significantly up-regulated phosphorylation sites were mainly involved in the key phosphoproteins regulating the insulin-related pathways, such as PI3K-AKT and RAS-MAPK pathways. Therefore, the MaSR strategy exhibits as a promising way in elucidating the biological processes with significant dysregulations.

Magnetic nanoparticles functionalized with maltosylated glycopeptide dendrimers were prepared via azide-alkynyl click reaction. The functionalized magnetic nanoparticles exhibited high hydrophilicity and good efficiency in glycopeptide enrichment by HILIC.

Stable isotope dimethyl labeling, a widely used method for quantitative proteomics, was extended to five channels for the first time. Comprehensive proteome and phosphoproteome quantification validated the high quantification accuracy and throughput of this five-plex method.

The titanium silicalite-1 (TS-1) with post-treatment of chemical selective desilication (T-TS-1) has been synthesized, characterized and applied as a potential adsorbent for selective capture of phosphopeptides from complex biological samples prior to mass spectrometry analysis. A T-TS-1 material-based successive phosphopeptide enrichment strategy has also been established for real biological sample analysis, which exhibited high enrichment selectivity and is comparable to existing materials.

We develop an acidic vapor assisted electrospray ionization strategy within an enclosed electrospray ionization source to counteract the ion suppression effects caused by trifluoroacetic acid. The mass spectrometry signal intensity of intact proteins was improved 10 times and the number of valid signals for E. coli intact protein samples was improved 96% by using this strategy.