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Highly selective and efficient enrichment of trace phosphorylated proteins or peptides from complex biological samples is of profound significance for the discovery of disease biomarkers in biological systems. In this study, a novel immobilized metal affinity chromatography (IMAC) material has been synthesized to improve the enrichment specificity and sensitivity for phosphopeptides by introducing a titanium phosphate moiety on a multilayer polysaccharide (hyaluronate (HA) and chitosan (CS)) coated Fe

Straw incorporation exerts important roles on greenhouse gas emissions and soil carbon (C) sequestration. However, few studies have comprehensively assessed the effects of straw incorporation on net global warming potential (NGWP) considering both emissions of greenhouse gas (GHG) and accumulation of SOC in Northeast China, the most important agricultural regions across the nation. An ongoing straw incorporation experiment was initiated in a maize cropping system of Northeast China from 2010, including three treatments: no straw incorporation (CK), incorporation of maize straw at a low level of 4000 kg ha −1 (S 4 ), and at a high level of 8000 kg ha −1 (S 8 ). At 2015, the crop yield was significantly elevated (11% and 21% for S 4 and S 8 , respectively) by the straw additions resulting from improved soil fertility. During the growing season of 2015, we found that the straw additions did not affect seasonal patterns of the N 2 O and CH 4 , but changed their seasonal magnitudes. Averaged over this period, CH 4 flux was very low based on its GWP, and straw incorporation tended to reduce the CH 4 emissions (6.6, 3.6 and 4.3 kg CO 2 eq ha −1 for CK, S 4 and S 8 , respectively); while straw incorporation significantly enhanced N 2 O emissions with the value of 429, 590 and 746 kg CO 2 eq ha −1 for CK, S 4 and S 8 , respectively. On the other hand, a significantly linear relationship (SOC sequestration rate = 0.29 × annual C input–0.57, R 2 = 0.99, P 0.05) was detected between annual straw C input and SOC sequestration rate over the 5-year cycles, indicating the soil we studied was still not C-saturated at least during the experimental interval. More notably, the C conversion rate (29%, the slope of the equation) was generally larger than the values reported in other region of China. These results implied and confirmed that the soil of Northeast China possessed greater potentials to sequestrate external C. Taken together, the SOC accumulation accounted for major portion of the NGWP and counteracted the extra emissions of GHGs (492, −1183 and −3040 kg CO 2 eq ha −1 for NGWP of CK, S 4 and S 8 , respectively). Therefore, the agricultural practise of straw incoporation was strongly recommended to be adopted in Northeat China to improve the soil productivity and simultaneously mitigate the negative influence of GHG emissions.

The vinyl-functionalized hybrid monolithic columns (75 and 150 μm i.d.) were prepared via sol-gel chemistry of tetramethoxysilane (TMOS) and vinyltrimethoxysilane (VTMS). The content of accessible vinyl groups was further improved after the monolithic column was post-treated with vinyldimethylethoxysilane (VDMES). The surface properties of monolithic columns were tailored via thiol-ene click reaction by using 1-octadecanethiol, sodium 3-mercapto-1-propanesulfonate and 2,2′-(ethylenedioxy)diethanethiol/vinylphosphonic acid, respectively. The preparing octadecyl-functionalized monolithic columns were adopted for proteomics analysis in cLC–MS/MS. A 37-cm-long × 75-μm-i.d. monolithic column could identify 3918 unique peptides and 1067 unique proteins in the tryptic digest of proteins from HeLa cells. When a 90-cm-long × 75-μm-i.d. monolithic column was used, the numbers of unique peptides and proteins were increased by 82% and 32%, respectively. Furthermore, strong cation exchange (SCX) monolithic columns (4 cm in length × 150 μm i.d.) were also prepared and coupled with the 37-cm-long × 75-μm-i.d. octadecyl-functionalized monolithic column for two-dimensional SCX-RPLC–MS/MS analysis, which could identify 17114 unique peptides and 3211 unique proteins.

In this study, we developed a centrifugation assisted solid phase extraction (SPE) method for the selective enrichment of phosphopeptides using a new Ti4+-IMAC material synthesized in-house. This new material has the feature of big size and large specific surface area which makes it more suitable to enrich phosphopeptides in a SPE way. The spin tips loaded with the Ti4+-IMAC material were applied to enrich phosphopeptides from the complex protein digests. It was found that phosphopeptides can be specifically enriched from tryptic digest of bovine serum albumin and β-casein at a molar ratio up to 1000:1. And about 4700 unique phosphorylated peptides can be identified with the specificity as high as 99% from the tryptic digest of HeLa cell proteins. This tip was demonstrated to have good column-to-column reproducibility. Furthermore, it is fitted to analyze minute amount of sample. Compared with the conventional solution method, the SPE method facilitated the rapid and complete separation of the material with solution, which making it a time-saving and convenient method for phosphopeptide enrichment. Compared with the commercial TiO2 material, this new materials yielded much more phosphopeptide identifications and much higher enrichment specificity.

Monoclonal antibodies (mAbs), are one of the most important protein drugs have attracted increasing attention. However, the pharmacokinetics of mAbs has not been fully investigated due to the complexity of protein drugs. Traditonal immuno-based approaches can not recognize the proteoforms of mAbs because of the long development cycles, prohibitive cost, and interactions between different proteins. Therefore, reliable qualitative and quantitative analysis of the proteoforms of mAbs in biological samples is of crucial importance. Herein, a novel method was developed for absolute quantitation of mAbs and their glycoforms in complex biological samples such as serum and tissues. With the combination of HILIC enrichment and parallel reaction monitoring by high resolution mass spectrometry, most of the glycoforms can be accurately quantified at the fmol level through the use of the model mAb of bevacizumab. More importantly, the structural confirmation can be achieved simultaneously without the need for additional experiments. This strategy can be readily applied to the pharmacokinetic study of glycosylation modification and biomarker discovery for clinical applications.

Sialylation typically occurs at the terminal of glycans, and its aberration often correlates with diseases including neurological diseases and cancer. However, the analysis of glycoprotein sialylation in complex biological samples is still challenging due to their low abundance. Herein, a histidine-bonded silica (HBS) material with a hydrophilic interaction and switchable surface charge was fabricated to enrich sialylated glycopeptides (SGPs) from the digest of proteomics samples. High selectivity toward SGPs was obtained by combining the superior hydrophilicity and switchable-charge characteristics. During the enrichment of sialylated glycopeptides from bovine fetuin digest, seven glycopeptides were detected even at the ratio of 1:5000 with the nonsialylated glycopeptides, demonstrating the high specificity of SGP enrichment by using HBS material. Then, HBS material was further utilized to selectively enrich SGPs from the protein digest of human serum, and 487 glycosites were identified from only 2 μL of human serum; 92.0% of the glycopeptides contained at least one sialic acid, indicating good performance for SGP enrichment by using HBS material. Furthermore, the prepared HBS material also has great potential applications in the analysis of glycoprotein sialylation from other complex biological samples.

Temporal tyrosine phosphorylation (pTyr) plays a crucial role in numerous cellular functions. The characterization of the tyrosine phosphorylation states of cells is of great interest for understanding the underlying mechanisms. In this study, we developed sensitive and cost-effective methods for the assay of the global protein tyrosine phosphorylation in complex samples by using a novel engineered pTyr binding protein, Src SH2 domain triple-point mutant (Trm-SH2). Taking the advantage of the pan-specific interaction of Trm-SH2 to pTyr, a high throughput approach was developed to determine the total protein tyrosine phosphorylation level in a sample. This method allowed the detection of 0.025 ng of tyrosine phosphorylated proteins. The Trm-SH2 was further exploited to develop a method to profile the global tyrosine phosphorylation state. When this approach was applied to analyze the tyrosine phosphoproteome upon stimulation, distinct patterns were observed. This approach is readily used in many research and clinical fields for the analysis of tyrosine phosphorylated proteins in complex samples, including classifying aberrant phosphotyrosine-dependent signaling in cancer.

The diversity of O-linked glycan structures has drawn increasing attention due to its vital biological roles. However, intact O-glycopeptides with different glycans are typically not well elucidated using the current methods. In this work, an integrated strategy was developed for comprehensive analysis of O-GalNAc glycosylation by combining hydrophilic interaction chromatography (HILIC) tip enrichment, beam-type collision induced decomposition (beam-CID) detection, and in silico deglycosylation method for spectra interpretation. In this strategy, the intact O-GalNAc glycopeptides were selectively enriched and the original spectra obtained by time-of-flight (TOF)-CID were preprocessed using an in silico deglycosylation method, enabling direct searching without setting multiple glycosylation modifications, which could significantly decrease the search space. This strategy was applied to analyze the O-GalNAc glycoproteome of human serum, leading to identification of 407 intact O-GalNAc glycopeptides from 93 glycoproteins. About 81% of the glycopeptides contained at least one sialic acid, which could reveal the microheterogeneity of O-GalNAc glycosylation. Up until now, this is the largest data set of intact O-GalNAc glycoforms from complex biological samples at the proteome level. Furthermore, this method is readily applicable to study O-glycoform heterogeneity in other complex biological systems.

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.

Many important experiments in proteomics including protein digestion, enzyme substrate screening, enzymatic labeling, etc., involve the enzymatic reactions in a complex system where numerous substrates coexists with an enzyme. However, the enzyme kinetics in such a system remains unexplored and poorly understood. Herein, we derived and validated the kinetics equations for the enzymatic reactions in complex system. We developed an iteration approach to depict the enzymatic reactions in complex system. It was validated by 630 time-course points from 24 enzymatic reaction experiments and was demonstrated to be a powerful tool to simulate the reactions in the complex system. By applying this approach, we found that the ratio of substrate depletion is independent of other coexisted substrates under specific condition. This observation was then validated by experiments. Based on this striking observation, a simplified model was developed to determine the catalytic efficiencies of numerous competing substrates presented in the complex enzyme reaction system. When coupled with high-throughput quantitative proteomics technique, this simplified model enabled the accurate determination of catalytic efficiencies for 2369 peptide substrates of a protease by using only one enzymatic reaction experiment. Thus, this study provided, in the first time, a validated model for the large scale determination of specificity constants which could enable the enzyme substrate screening approach turned from a qualitative method of identifying substrates to a quantitative method of identifying and prioritizing substrates. Data are available via ProteomeXchange with identifier PXD004665.

© 2016 Nature America, Inc. All rights reserved. We present a new strategy for systematic identification of phosphotyrosine (pTyr) by affinity purification mass spectrometry (AP-MS) using a Src homology 2 (SH2)-domain-derived pTyr superbinder as the affinity reagent. The superbinder allows for markedly deeper coverage of the Tyr phosphoproteome than anti-pTyr antibodies when an optimal amount is used. We identified â 1/420,000 distinct phosphotyrosyl peptides and >10,000 pTyr sites, of which 36% were 'novel', from nine human cell lines using the superbinder approach. Tyrosine kinases, SH2 domains and phosphotyrosine phosphatases were preferably phosphorylated, suggesting that the toolkit of kinase signaling is subject to intensive regulation by phosphorylation. Cell-type-specific global kinase activation patterns inferred from label-free quantitation of Tyr phosphorylation guided the design of experiments to inhibit cancer cell proliferation by blocking the highly activated tyrosine kinases. Therefore, the superbinder is a highly efficient and cost-effective alternative to conventional antibodies for systematic and quantitative characterization of the tyrosine phosphoproteome under normal or pathological conditions.

In recent years, polymerizations based on click reactions (thiol-ene, thiol-yne, thiol-Michael, thiol-epoxy and amine-epoxy) have been utilized to prepare monolithic columns. These polymerization systems are easily carried out under mild conditions. Either hybrid or organic monolithic columns fabricated by click polymerization demonstrated homogeneous network structures. For separation of small molecules, the column efficiencies, such as plate height with less than 10 µm, have been greatly improved comparing with organic monolithic columns prepared with free radical polymerization. In this review, we will summarize recent progress on the preparation of monolithic columns and their chromatographic performances.

A facile approach was exploited for fast preparation of polymer-based monoliths in UV-transparent fused-silica capillaries via "one-pot" photo-initiated thiol-acrylate polymerization reaction of dipentaerythritolpenta-/hexaacrylate (DPEPA) and 1-octadecanethiol (ODT) in the presence of porogenic solvents (1-butanol and ethylene glycol). Due to relative insensitivity of oxygen inhibition in thiol-ene free-radical polymerization, the polymerization could be performed within 5 min. The effects of composition of prepolymerization solution on the morphology and permeability of poly(ODT-co-DPEPA) monoliths were investigated in detail by adjusting the content of monomer and binary porogen ratio. The physical properties of poly(ODT-co-DPEPA) monoliths were characterized by Fourier transform infrared spectroscopy (FT-IR), mercury intrusion porosimetry (MIP) and nitrogen adsorption/desorption measurement. The evaluation of chromatographic performance was carried out by capillary liquid chromatography (cLC). The results indicated that the poly(ODT-co-DPEPA) monolith was homogeneous and permeable, and also possessed a typical reversed-phase retention mechanism in cLC with high efficiency (∼75,000 N m(-1)) for separation of alkylbenzenes. Eventually, the further separation of tryptic digest of proteins by cLC tandem mass spectrometry (cLC-MS/MS) demonstrated its potential in the analysis of biological samples.

Large-scale N-glycoproteome studies have been hindered by poor solubility of hydrophobic membrane proteins and the complexity of proteome samples. Herein, we developed a detergent-assisted glycoprotein capture method to reduce these issues by conducting hydrazide chemistry-based glycoprotein capture in the presence of strong detergents such as sodium dodecyl sulfate and Triton X-100. The strong detergents helped to solubilize hydrophobic membrane proteins and then increased the access of hydrazide groups to oxidized glycoproteins, thus increasing the coverage of the N-glycoproteome. Compared with the conventional glycopeptide capture method, the detergent-assisted glycoprotein capture approach nearly doubled the number of N-glycosylation sites identified from HEK 293T cells with improved specificity. Application of this approach in the larger scale N-glycoproteomics analysis of the HEK 293T cell membrane led to the identification of 2253 unique N-glycosites from 953 proteins. Furthermore, the application of this approach to human serum resulted in the identification of 850 N-glycosylation sites without any immunodepletion or fractionation. Overall, the detergent-assisted glycoprotein capture method simplified the capture process, and it increased the number of sites observed on both hydrophobic membrane proteins and hydrophilic secreted proteins.

A hybrid fluorous monolithic column was simply prepared via photo-initiated free radical polymerization of an acrylopropyl polyhedral oligomeric silsesquioxane (acryl-POSS) and a perfluorous monomer (2,2,3,3,4,4,5,5,6,6,7,7-dodecafluoroheptyl acrylate) in UV-transparent fused-silica capillaries within 5min. The physical characterization of hybrid fluorous monolith, including scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, mercury intrusion porosimetry (MIP) and nitrogen adsorption/desorption measurement was performed. Chromatographic performance was also evaluated by capillary liquid chromatography (cLC). Due to the fluorous-fluorous interaction between fluorous monolith and analytes, fluorobenzenes could well be separated, and the column efficiencies reached 86,600-92,500plates/m at the velocity of 0.87mm/s for alkylbenzenes and 51,900-76,000plates/m at the velocity of 1.10mm/s for fluorobenzenes. Meanwhile, an approach to integrate nanoelectrospray ionization (ESI) emitter with hybrid fluorous monolithic column was developed for quantitative determination of perfluoroalkyl acids by nanoHPLC-ESI-MS/MS. The integration design could minimize extracolumn volume, thus excluding undesirable peak broadening and improving separation performance.

A novel silica-based stationary phase with branched octadecyl groups was prepared by the sequential employment of the Michael addition reaction and photoinduced thiol-yne click chemistry with 3-aminopropyl-functionalized silica microspheres as the initial material. The resulting stationary phase denoted as SiO2 -N(C18)4 was characterized by elemental analysis, FTIR spectroscopy and Raman spectroscopy, demonstrating the existence of branched octadecyl groups in silica microspheres. The separations of benzene homologous compounds, acid compounds and amine analogues were conducted, demonstrating mixed-mode separation mechanism on SiO2 -N(C18)4 . Baseline separation of basic drugs mixture was acquired with the mobile phase of acetonitrile/H2 O (5%, v/v). SiO2 -N(C18)4 was further applied to separate Corydalis yanhusuo Wang water extracts, and more baseline separation peaks were obtained for SiO2 -N(C18)4 than those on Atlantis dC18 column. It can be expected that this new silica-based stationary phase will exhibit great potential in the analysis of basic compounds.

Cyclodextrins and their derivatives are one of the most common and successful chiral selectors. However, there have been few publications about the use of cyclodextrin-modified monoliths. In this study, organic hybrid monoliths were prepared by the immobilization of derivatized β-cyclodextrin alone or with l-2-allylglycine hydrochloride to the polyhedral oligomeric silsesquioxane methacryl substituted monolith. The main topic of this study is a combined system with dual chiral selectors (l-2-allylglycine hydrochloride and β-cyclodextrin) as monolithic chiral stationary phase. The effect of l-2-allylglycine hydrochloride concentration on enantioseparation was investigated. The enantioseparation of the four acidic compounds with resolutions up to 2.87 was achieved within 2.5 min on the prepared chiral monolithic column in capillary liquid chromatography. Moreover, the possible mechanism of enantioseparation was discussed.

Micropattern topography is widely investigated for its role in mediating stem cell differentiation, but remains unexplored for phenotype switch between mature cell types. This study investigated the potential of inducing tenogenic phenotype in human dermal fibroblasts (hDFs) by artificial elongation of cultured cells. Our results showed that a parallel microgrooved topography could convert spread hDFs into an elongated shape and induce a predominant tenogenic phenotype as the expression of biomarkers was significantly enhanced, such as scleraxis, tenomodulin, collagens I, III, VI, and decorin. It also enhanced the expression of transforming growth factor (TGF)-β1, but not α-smooth muscle actin. Elongated hDFs failed to induce other phenotypes, such as adiopogenic, chondrogenic, neurogenic, and myogenic lineages. By contrast, no tenogenic phenotype could be induced in elongated human chondrocytes, although chondrogenic phenotype was inhibited. Exogenous TGF-β1 could enhance the tenogenic phenotype in elongated hDFs at low dose (2 ng/ml), but promoted myofibroblast transdifferentiation of hDFs at high dose (10 ng/ml), regardless of cell shape. Elongated shape also resulted in decreased RhoA activity and increased Rho-associated protein kinase (ROCK) activity. Antagonizing TGF-β or inhibiting ROCK activity with Y27632 or depolymerizing actin with cytochalasin D could all significantly inhibit tenogenic phenotype induction, particularly in elongated hDFs. In conclusion, elongation of cultured dermal fibroblasts can induce a predominant tenogenic phenotype likely via synergistic effect of TGF-β and cytoskeletal signaling.

The high accumulation of multiwalled carbon nanotubes (MWCNTs) in the liver results in their internalization in hepatic cells and perturbations to CYP enzymes, including CYP3A4. Surface chemical modifications on MWCNTs cause enhancement, inhibition, or no effect in CYP3A4 activity. Computational analysis identifies chemical structures on the surface modifications that induce CYP3A4 inhibition or prevent its perturbation.

Selective enrichment of specific peptides is an effective way to identify low abundance proteins. Fractionation of peptides prior to mass spectrometry is another widely used approach to reduce sample complexity in order to improve proteome coverage.In this study, we designed a multi-stage digestion strategy to generate peptides with different trypsin cleavage kinetics. It was found that each of the collected peptide fractions yielded many new protein identifications compared to the control group due to the reduced complexity. The overlapping peptides identified between adjacent fractions were very low, indicating that each fraction had different sets of peptides. The multi-stage digestion strategy separates tryptic peptides with different cleavage kinetics while RPLC separates peptides with different hydrophobicity. These two separation strategies were highly orthogonal, and showed an effective multidimensional separation to improve proteome coverage.

Selective enrichment of glycopeptides from complex sample followed by cleavage of N-glycans by PNGase F to expose an easily detectable mark on the former glycosylation sites has become the popular protocol for comprehensive glycoproteome analysis. On account of the high enrichment specificity, hydrazide chemistry based solid-phase extraction of N-linked glycopeptides technique has sparked numerous interests. However, the enzymatic release of glycopeptides captured by hydrazide beads through direct incubation of the beads with PNGase F is not efficient due to the inherent steric hindrance effect. In this study, we developed a hydroxylamine assisted PNGase F deglycosylation (HAPD) method using the hydroxylamine to release glycopeptides captured on the hydrazide beads through the cleavage of hydrazone bonds by transamination followed with the PNGase F deglycosylation of the released glycopeptides in the free solution. Because of the homogeneous condition for the deglycosylation, the recovery of deglycosylated peptides (deglycopeptides) was improved significantly. It was found that 27% more N-glycosylation sites were identified by the HAPD strategy compared with the conventional method. Moreover, the ratio of identified N-terminal glycosylated peptides was improved over 5-fold.

Polyacrylamide gel electrophoresis (PAGE) is a powerful protein separation technology. Combined with mass spectrometry, it could identify thousands of proteins in proteomics analysis. However, the time-consuming procedure restricts its broad applications in proteomics study. In this work, it was found that high concentration of trypsin did not compromise subsequent phosphopeptide enrichment after in-gel digestion, but could promote the in-gel digestion. Hence, a new, fast and robust digestion method was established. Firstly, 50 μg of HeLa cell protein was separated into 5 fractions by SDS-PAGE, and then the proteins were digested with high concentration trypsin for 30 min. Finally, the phosphopeptides were enriched by Ti-IMAC Tip. After liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis, about 2000 phosphorylation sites were identified in the experimental group, while less than 1500 phosphorylation sites were identified in control group. With the aid of high concentration of trypsin, in-gel digestion could be completed within only 30 min, and more phosphorylation site identifications and lower percentage of missed cleavages were acquired than those in control experiments. The experiment results demonstrated that high concentration of trypsin could not only accelerate the in-gel digestion, but also improve the phosphoproteome coverage.

Two kinds of hybrid monoliths were first prepared via thiol-epoxy click polymerization using a multi-epoxy monomer, octaglycidyldimethylsilyl POSS (POSS-epoxy), and two multi-thiols, trimethylolpropanetris(3-mercaptopropionate) (TPTM) and pentaerythritoltetrakis(3-mercaptopropionate) (PTM), respectively, as the precursors. The resulting two hybrid monoliths (assigned as POSS-epoxy-TPTM and POSS-epoxy-PTM) not only possessed high thermal, mechanical and chemical stabilities, but also exhibited well-controlled 3D skeletal microstructure and high efficiency in capillary liquid chromatography (cLC) separation of small molecules. The highest column efficiency reached 182,700N/m (for butylbenzene) on the monolith POSS-epoxy-PTM at the velocity of 0.75mm/s. Furthermore, the hybrid monolith POSS-epoxy-PTM was successfully applied for cLC separations of various samples, not only standard compounds such as alkylbenzenes, PAHs, phenols and dipeptides, as well as intact proteins, but also complex samples of EPA 610 and BSA digest.

Monolithic poly(methacrylate epoxy cyclosiloxane-co-polyhedral oligomeric silsesquioxanes) (epoxy-MA–POSS) capillary columns have been prepared via either photo- or thermally-initiated polymerization of the corresponding monomers using a 1-propanol/PEG 400 mixture as porogens. Photochemical polymerization was accomplished by irradiation of the UV-transparent capillary for 10 min at room temperature, while thermal polymerization was performed at 55 °C, 60 °C or 65 °C for 18 h. The evaluation of chromatographic property for two hybrid epoxy-MA–POSS monoliths was carried out. The results indicate that hybrid monoliths fabricated by photochemical initiation exhibit higher column efficiency (97,000–98,400 plates/m) than those synthesized by thermal polymerization (41,100–48,000 plates/m) in cLC. The higher efficiency of photo-initiated hybrid monoliths is closely related to lower eddy dispersion (A-term) and mass transfer resistance (C-term).

A novel "one-pot" approach was developed for ultrarapid preparation of various hybrid monolithic columns in UV-transparent fused-silica capillaries via photoinitiated thiol-acrylate polymerization of an acrylopropyl polyhedral oligomertic silsesquioxane (acryl-POSS) and a monothiol monomer (1-octadecanethiol or sodium 3-mercapto-1-propanesulfonate) within 5 min, in which the acrylate not only homopolymerizes, but also couples with the thiol. This unique combination of two types of free-radical reaction mechanisms offers a simple way to fabricate various acrylate-based hybrid monoliths. The physical characterization, including scanning electron microscopy (SEM), Fourier transform infrared (FT-IR) spectroscopy, and thermal gravimetric analysis was performed. The results indicated that the monothiol monomers were successfully incorporated into acryl-POSS-based hybrid monoliths. The column efficiencies for alkylbenzenes on the C18-functionalized hybrid monolithic column reached to 60 000-73 500 plates/m at the velocity of 0.33 mm/s in capillary liquid chromatography, which was far higher than that of previously reported POSS-based columns prepared via thermal-initiated free-radical polymerization without adding any thiol monomers. By plotting the plate height (H) of the alkylbenzenes versus the linear velocity (u) of the mobile phase, the results revealed a retention-independent efficient performance of small molecules in the isocratic elution. These results indicated that more homogeneous hybrid monoliths formed via photoinitiated thiol-acrylate polymerization; particularly, the use of the multifunctional cross-linker possibly prevented the generation of gel-like micropores, reducing mass transfer resistance (C-term). Another sulfonate-containing hybrid monolithic column also exhibited hydrophobicity and ion-exchange mechanism, and the dynamic binding capacity was calculated as 71.1 ng/cm (75 μm i.d.).

The marine microalga Tetraselmis subcordiformis could photoproduce hydrogen under the regulation of carbonyl cyanide m-chlorophenylhydrazone (CCCP), and a hydrogen production process kinetic analysis was characterized by two peaks, suggesting that two distinct mechanisms might exist in this alga. Therefore, 2D nanoliquid chromatography-tandem mass spectrometry (LC-MS/MS) was introduced to analyze the proteome of samples from different time points. A total of 912 proteins were identified, providing a global view of the cellular responses at the proteomic level. These proteins can be divided into multiple functional groups including stress responses, energy metabolism and redox homeostasis. The quantitative proteomic data provided more details on the electron donors for hydrogen production. During the first stage, photosystem II produced electrons for hydrogen production; during the second stage, metabolites were the major electron donors via nonphotochemical plastoquinone reduction by NADH dehydrogenase.

Low column efficiency for small molecules in reversed-phase chromatography is a major problem commonly encountered in polymer-based monoliths. Herein, a novel highly crosslinked porous polymeric monolith was in situ prepared by using a multi-acrylate monomer, dipentaerythritol penta-/hexa-acrylate (DPEPA), as crosslinker, which copolymerized with lauryl methacrylate (LMA) as functional monomer in a UV-transparent fused-silica capillary via photo-initiated free-radical polymerization within 5 min. The mechanical stability and permeability of the resulting poly(LMA- co -DPEPA) monolith were characterized in detail. One series of highly crosslinked poly(LMA- co -DPEPA) columns were prepared with relatively higher content of crosslinker (63.3%) in the precursor. Although they exhibited lower permeability, high column efficiency for alkylbenzenes was acquired in cLC, and the minimum plate height (column B) was in the range of 6.04–9.00 μm, corresponding to 111,000–165,000 N m −1 . Meanwhile, another series of poly(LMA- co -DPEPA) columns prepared with relatively lower content of crosslinker (52.7%) in the precursor exhibited higher permeability, but the minimum plate height (column E) was relatively low in the range of 10.75–20.04 μm for alkylbenzenes, corresponding to 50,000–93,000 N m −1 . Compared with common poly(LMA- co -EDMA) columns previously reported, the highly crosslinked poly(LMA- co -DPEPA) columns using a multi-acrylate monomer as crosslinker possessed remarkably high column efficiency for small molecules in cLC. By plotting of plate height ( H ) of alkylbenzenes versus the linear velocity ( u ) of mobile phase, the results revealed a retention-independent efficient performance of small molecules in the isocratic elution, indicating that the use of multi-functional crosslinker possibly prevents the generation of gel-like micropores in the poly(LMA- co -DPEPA) monolith, reducing the mass transfer resistance ( C -term).

A facile template-free strategy for the synthesis of mesoporous phenolic polymers with attractive porosities, nitrogen-containing functionalities, and intrinsic hydrophilic skeletons is presented. The resultant polymer has a high BET surface area (548 m2 g–1) and mesopore size (13 nm) and exhibits superior glycopeptide-capturing performance, thus, revealing the potential application of mesoporous polymers in highly selective glycopeptide enrichment. This general capture protocol may open up new opportunities for the development of glycoproteomes.

In consideration of the intrinsic complexity of cancer, just being a delivery nanovehicle for the nanocarrier is no longer enough to fulfill requirements of dealing with cancer. In this regard, the multifunctional nanocarrier appears to be an appealing choice in cancer treatment. Herein, the novel multifunctional nanocarrier (Fe3O4-NS-C3N4@mSiO2-PEG-RGD) possessing properties of dual targeting (the peptide- and magnetism-mediated targeting), imaging (one- and two-photon modes), pH-triggered release of loaded anticancer drug, and synergistic treatment (photodynamic therapy (PDT) combined with chemotherapy) are successfully developed. The nanocarrier specifically centralizes within cancer cells with the enhanced amount through the dual targeting ability and is facilely tracked under one- and two-photon imaging modes attributed to the autofluorescence. Then, visible light irradiation-induced PDT combined with low pH-triggered chemotherapy synergistically cooperate to efficiently kill cancer cells. Following the above process, the multifunctional nanocarrier demonstrates effective inhibition of the growth of A549 and HeLa cancer cells. The efficient manipulation of Fe3O4-NS-C3N4@mSiO2-PEG-RGD also implies potential applications of the multifunctional nanocarrier in delivery of different agents. Furthermore, it might also broaden the scope of fabrication of the multifunctional nanocarrier for inhibiting the growth of cancer cells.

Lipid droplets (LDs) are ubiquitous organelles that serve as a neutral lipid reservoir and a hub for lipid metabolism. Manipulating LD formation, evolution, and mobilization in oleaginous species may lead to the production of fatty acid-derived biofuels and chemicals. However, key factors regulating LD dynamics remain poorly characterized. Here we purified the LDs and identified LD-associated proteins from cells of the lipid-producing yeast Rhodosporidium toruloides cultured under nutrient-rich, nitrogen-limited, and phosphorus-limited conditions. The LD proteome consisted of 226 proteins, many of which are involved in lipid metabolism and LD formation and evolution. Further analysis of our previous comparative transcriptome and proteome data sets indicated that the transcription level of 85 genes and protein abundance of 77 proteins changed under nutrient-limited conditions. Such changes were highly relevant to lipid accumulation and partially confirmed by reverse transcription-quantitative PCR. We demonstrated that the major LD structure protein Ldp1 is an LD marker protein being upregulated in lipid-rich cells. When overexpressed in Saccharomyces cerevisiae , Ldp1 localized on the LD surface and facilitated giant LD formation, suggesting that Ldp1 plays an important role in controlling LD dynamics. Our results significantly advance the understanding of the molecular basis of lipid overproduction and storage in oleaginous yeasts and will be valuable for the development of superior lipid producers.

A facile approach was developed for direct preparation of organic monoliths via the alkaline-catalyzed thiol-epoxy click polymerization. Two organic monoliths were prepared by using tetraphenylolethane glycidyl ether as a multiepoxy monomer, and trimethylolpropane tris(3-mercaptopropionate) and pentaerythritol tetrakis(3-mercaptopropionate) as the multithiol monomer, respectively, in the presence of a ternary porogenic system (DMSO/PEG200/H2O). The obtained organic monoliths showed high thermal, mechanical and chemical stabilites. Benefiting from the step-growth polymerization process, two organic monoliths possessed well-defined 3D framework microstructure, and exhibited high permeabilities and column efficiencies in capillary liquid chromatography. A series of neutral, basic and acidic small molecules were used to comprehensively evaluate the separation abilities of these monoliths, and satisfactory chromatographic performance with column efficiencies ranged from 35,500 to 132,200 N/m was achieved, demonstrating good separation abilities of these organic monoliths prepared via thiol-epoxy click polymerization approach. Besides, multiple retention mechanisms, including hydrophobic, hydrophilic and π-π conjugate interactions were observed during the separation of analytes on these monoliths, which would make them promising for more extensive applications in capillary liquid chromatography.

Highly selective and efficient enrichment of glycans from complex biological samples is of great significance for the discovery and diagnosis of disease via identifying the related biomarkers. Mesoporous carbon materials were widely employed for the selective enrichment of glycans due to the strong interactions between carbon and glycans. In this study, novel carbon-functionalized ordered magnetic mesoporous silica composites (denoted as Fe3O4@3SiO2@mSiO2–C) with a core–shell structure, high carbon content, excellent hydrophilic property and unique magnetic character were designed and synthesized. A strategy involving CTAB as the mesoporous structure-directing agent and the carbon precursor during in situ carbonization was proposed. Besides, a compact silica layer with adequate thickness was essential to protect the magnetic core during the sulphonation process in further high-temperature calcination. As a result, the obtained Fe3O4@3SiO2@mSiO2–C composites exhibited a high carbon content (25%) with graphite structure, rapid magnetic separation (within 10 s), large pore volume (0.257 m3 g−1), high BET surface area (269.14 m2 g−1) and a well-ordered mesostructure (3.39 nm). In addition, a strong magnetic response with a saturation magnetization value (59.8 emu g−1) has been confirmed. By taking the advantage of the special interaction between the carbon and glycans, the size-exclusion ability and highly hydrophilic as well as unique magnetic properties, the Fe3O4@3SiO2@mSiO2–C composites exhibit satisfying enrichment ability in glycomic analysis. Better selectivity and efficiency than active carbon have been confirmed. Furthermore, 42 N-linked glycans with sufficient peak intensities were obtained from human serum after treatment with the Fe3O4@3SiO2@mSiO2–C composites, showing great potential as a tool for the enrichment and detection of glycans in biological samples.

A facile approach based on thiol-methacrylate Michael addition click reaction was developed for construction of porous hybrid monolithic materials. Three hybrid monoliths were prepared via thiol-methacrylate click polymerization by using methacrylate-polyhedral oligomeric silsesquioxane (POSS) (cage mixture, n = 8, 10, 12, POSS-MA) and three multi-thiol crosslinkers, 1,6-hexanedithiol (HDT), trimethylolpropane tris(3-mercaptopropionate) (TPTM) and pentaerythritol tetrakis(3-mercaptopropionate) (PTM), respectively, in the presence of porogenic solvents ( n -propanol and PEG 200) and a catalyst (dimethylphenylphosphine, DMPP). The obtained monoliths possessed high thermal and chemical stabilities. Besides, they all exhibited high column efficiencies and excellent separation abilities in capillary liquid chromatography (cLC). The highest column efficiency could reach ca. 195,000 N/m for butylbenzene on the monolith prepared with POSS-MA and TPTM (monolith POSS-TPTM) in reversed-phase (RP) mode at 0.64 mm/s. Good chromatographic performance were all achieved in the separations of polycyclic aromatic hydrocarbons (PAHs), phenols, anilines, EPA 610 as well as bovine serum albumin (BSA) digest. The high column efficiencies in the range of 51,400–117,000 N/m (achieved on the monolith POSS-PTM in RP mode) convincingly demonstrated the high separation abilities of these thiol-methacrylate based hybrid monoliths. All the results demonstrated the feasibility of the phosphines catalyzed thiol-methacrylate Michael addition click reaction in fabrication of monolithic columns with high efficiency for cLC applications.

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.

The highly selective and efficient capture of heterogeneous types of phosphopeptides is critical for comprehensive and in-depth phosphoproteome analysis, but it still remains a challenge since the lack of affinity material with large binding capacity and controllable specificity. Here, a new affinity material was prepared to improve the enrichment capacity and endue the tunable specificity by introducing guanidyl onto poly(glycidyl methacrylate) (PGMA) modified Fe3O4 microsphere (denoted as Fe3O4@PGMA-Guanidyl). The thick polymer shell endows the composite microsphere with large amount of guanidyl and is beneficial to enhancing the affinity interaction between phosphopeptides and the material. Interestingly, the Fe3O4@PGMA-Guanidyl possesses tunable enriching ability for global phosphopeptides or only multiphosphopeptides through simple regulation of buffer composition. The composite has large enrichment capacity (200 mg g(-1)), extremely high detection sensitivity (0.5 fmol), high enrichment recovery (91.30%), great specificity, and rapid magnetic separation. Moreover, the result of the application to capture of phosphopeptides from tryptic digest of nonfat milk has demonstrated the great potential of Fe3O4@PGMA-Guanidyl in detection and identification of low-abundance phosphopeptides of interest in biological sample.

Comprehensive analysis of the post-translational modifications (PTMs) on proteins at proteome level is crucial to elucidate the regulatory mechanisms of various biological processes. In the past decades, thanks to the development of specific PTM enrichment techniques and efficient multidimensional liquid chromatography (LC) separation strategy, the identification of protein PTMs have made tremendous progress. A huge number of modification sites for some major protein PTMs have been identified by proteomics analysis. In this review, we first introduced the recent progresses of PTM enrichment methods for the analysis of several major PTMs including phosphorylation, glycosylation, ubiquitination, acetylation, methylation, and oxidation/reduction status. We then briefly summarized the challenges for PTM enrichment. Finally, we introduced the fractionation and separation techniques for efficient separation of PTM peptides in large-scale PTM analysis.

Albeit much less abundant than Ser/Thr phosphorylation (pSer/pThr), Tyr phosphorylation (pTyr) is considered as a hallmark in cellular signal transduction. However, its analysis at the proteome level remains challenging. The conventional immunopurification (IP) approach using antibodies specific to pTyr sites is known to have low sensitivity, poor reproducibility and high cost. Our recent study indicated that SH2 domain-derived pTyr-superbinder is a good replacement of pTyr antibody for the specific enrichment of pTyr peptides for phosphoproteomics analysis. In this study, we presented an efficient SH2 superbinder based workflow for the sensitive analysis of tyrosine phosphoproteome. This new method can identify 41% more pTyr peptides than the previous method. Its excellent performance was demonstrated by the analysis of a variety of different samples. For the highly tyrosine phosphorylated sample, for example, pervanadate-treated Jurkat T cells, it identified over 1800 high confident pTyr sites from only 2 mg of proteins. For the unstimulated Jurkat cells, where the pTyr events rarely occurred, it identified 343 high confident pTyr sites from 5 mg of proteins, which was 31% more than that obtained by the antibody-based method. For the heterogeneous sample of tissue, it identified 197 high confident pTyr sites from 5 mg protein digest of mouse skeletal muscle. In general, it is a sensitive, robust and cost-effective approach and would have wide applications in the study of the regulatory role of tyrosine phosphorylation in diverse physiological and pathological processes.

The circadian clock is an endogenous oscillator that drives daily rhythms in physiology, behavior and gene expression. The underlying mechanisms of circadian timekeeping are cell-autonomous and involve oscillatory expression of core clock genes that is driven by interconnecting transcription-translation feedback loops (TTFLs). Circadian clock TTFLs are further regulated by post-translational modifications, in particular phosphorylation. The hippocampus plays an important role in spatial memory and the conversion of short-term to long-term memory. Several studies have reported the presence of a peripheral oscillator in the hippocampus, and have highlighted the importance of circadian regulation in memory formation. Given the general importance of phosphorylation in circadian clock regulation, we performed global quantitative proteome and phosphoproteome analyses of the murine hippocampus across the circadian cycle, applying spiked-in labeled reference and high accuracy mass spectrometry. Of the 3052 proteins and 2868 phosphopeptides on 1368 proteins that were accurately quantified, 1.7% of proteins and 5.2% of phosphopeptides exhibited time-of-day-dependent expression profiles. The majority of circadian phosphopeptides displayed abrupt fluctuations at mid-to-late day without underlying rhythms of protein abundance. Bioinformatic analysis of cyclic phosphorylation events revealed their diverse distribution in different biological pathways, most notably, cytoskeletal organization and neuronal morphogenesis. This study provides the first large-scale, quantitative mass spectrometry analysis of the circadian phosphoproteome and proteome of the murine hippocampus, and highlights the significance of rhythmic regulation at the post-translational level in this peripheral oscillator. In addition to providing molecular insights into the hippocampal circadian clock, our results will assist in the understanding of genetic factors that underlie rhythms-associated pathological states of the hippocampus.

Tyrosine phosphorylation (pTyr) is important for normal physiology and implicated in many human diseases, particularly cancer. Identification of pTyr sites is critical to dissecting signaling pathways and understanding disease pathologies. However, compared with serine/threonine phosphorylation (pSer/pThr), the analysis of pTyr at the proteome level is more challenging due to its low abundance. Here, we developed a biphasic affinity chromatographic approach where Src SH2 superbinder was coupled with NeutrAvidin affinity chromatography, for tyrosine phosphoproteome analysis. With the use of competitive elution agent biotin-pYEEI, this strategy can distinguish high-affinity phosphotyrosyl peptides from low-affinity ones, while the excess competitive agent is readily removed by using NeutrAvidin agarose resin in an integrated tip system. The excellent performance of this system was demonstrated by analyzing tyrosine phosphoproteome of Jurkat cells from which 3,480 unique pTyr sites were identified. The biphasic affinity chromatography method for deep Tyr phosphoproteome analysis is rapid, sensitive, robust, and cost-effective. It is widely applicable to the global analysis of the tyrosine phosphoproteome associated with tyrosine kinase signal transduction.

Two monolithic polymer columns were directly prepared in the UV-transparent fused-silica capillaries via photoinitiated thiol-yne click polymerization of 1,7-octadiyne (ODY) with a dithiol (1,6-hexanedithiol, 2SH) or a tetrathiol (pentaerythriol tetrakis(3-mercaptopropionate), 4SH) within 15 min. The rapid polymerization provided a time-saving approach to optimize preparation conditions. Then, two porogenic systems of diethylene glycol diethyl ether (DEGDE)/tetrahydrofuran (THF) and DEGDE/poly(ethylene glycol) (PEG, Mn = 200) were found to effectively control the porous structure of two kinds of polymeric monoliths (O2SH and O4SH), respectively. The almost disappearance of thiol and alkynyl vibrations (2560 and 2115 cm(-1), respectively) in infrared spectra and Raman spectra indicated a high conversion of the thiol-yne polymerization reaction. The thiol-yne polymerization was further proved by analyzing the energy-dispersive X-ray spectrum (EDS), MALDI-TOF mass spectrum, and elemental data. Scanning electron microscopy (SEM) images showed the monolithic polymer columns with homogeneous porous structure and macropore size of 0.5-1.0 μm, which facilitated the minimum plate heights of 10.0-12.0 μm for alkylbenzenes in reversed-phase liquid chromatography (RPLC). The low values of the A and C terms (1.0 μm and15.5 ms, respectively) in the van Deemter equation were similar to those obtained by some monolithic silica columns. The BSA tryptic digest was also separated on the monolithic polymer column by cLC-MS/MS. The result with 85% protein coverage was better than those given by some hybrid monolithic columns. The monolithic polymer columns were further applied for separation of phenols, natural products, and standard proteins and demonstrated satisfactory separation ability.

As an attractive alternative to organic and silica monoliths, hybrid organic–inorganic monolith somewhat combines the advantages of them, such as high surface area, excellent mechanical strength, and thermal stability. We have reviewed the preparation and application of hybrid monoliths in 2011 and 2013. The preparation approaches have been mainly summarized into three categories: (1) common sol–gel process using trialkoxysilanes and tetraalkoxysilanes as precursors; (2) “one-pot” process of alkoxysilanes and organic monomers simultaneously via sol–gel chemistry and free radical polymerization; and (3) other polymerization of silane-containing monomers. Herein, we would focus on the recent progress and development of preparation approaches, mainly covering the literatures since July of 2012. First, the direct synthesis approach of hybrid monoliths via sol–gel chemistry and following postmodification was an important route to fabricate various monolithic stationary phases, particularly, to modify the hybrid monoliths containing amino, epoxy, vinyl, and other groups. Second, “one-pot” process, as a novel preparation approach of hybrid monoliths, was further developed in the past 2 years, in which various organic functional monomers, not only water-soluble monomers, but also hydrophobic monomers could be added in the preparation system. Other polymerization techniques in the preparation of organic monolithic materials, particularly, free radical polymerization and ring-opening polymerization, were successfully transferred to fabricate the hybrid monoliths by using silane-containing monomers including POSS monomers or other self-synthesized monomers.

Two kinds of hybrid monolithic columns were prepared by using methacrylate epoxy cyclosiloxane (epoxy-MA) as functional monomer, containing three epoxy moieties and one methacrylate group. One column was in situ fabricated by ring-opening polymerization of epoxy-MA and 1,10-diaminodecane (DAD) using a porogenic system consisting of isopropanol (IPA), H2O and ethanol at 65°C for 12h. The other was prepared by free radical polymerization of epoxy-MA and ethylene dimethacrylate (EDMA) using 1-propanol and 1,4-butanediol as the porogenic solvents at 60°C for 12h. Two hybrid monoliths were investigated on the morphology and chromatographic assessment. Although two kinds of monolithic columns were prepared with epoxy-MA, their morphologies looked rather different. It could be found that the epoxy-MA-DAD monolith possessed higher column efficiencies (25,000-34,000plates/m) for the separation of alkylbenzenes than the epoxy-MA-EDMA monolith (12,000-13,000plates/m) in reversed-phase nano-liquid chromatography (nano-LC). Depending on the remaining epoxy or methacrylate groups on the surface of two pristine monoliths, the epoxy-MA-EDMA monolith could be easily modified with 1-octadecylamine (ODA) via ring-opening reaction, while the epoxy-MA-DAD monolith could be modified with stearyl methacrylate (SMA) via free radical reaction. The chromatographic performance for the separation of alkylbenzenes on SMA-modified epoxy-MA-DAD monolith was remarkably improved (42,000-54,000 plates/m) when compared with that on pristine epoxy-MA-DAD monolith, while it was not obviously enhanced on ODA-modified epoxy-MA-EDMA monolith when compared with that on pristine epoxy-MA-EDMA monolith. The enhancement of the column efficiency of epoxy-MA-DAD monolith after modification might be ascribed to the decreased mass-transfer resistence. The two kinds of hybrid monoliths were also applied for separations of six phenols and seven basic compounds in nano-LC.

Microgravity and simulated microgravity (SMG) have quite significant effects on numerous microbial cellular processes. The effects of SMG on the production of recombinant proteins and transcription profiling in prokaryotic and eukaryotic expression host have been investigated. The present study showed that SMG significantly enhanced the specific productivities and activities of the reporter enzymes PGUS and AtXYN that were expressed in recombinant Pichia pastoris. Proteomic profiling revealed that 21 proteins were significantly up-regulated and 35 proteins were drastically down-regulated at the stationary phase, when the recombinant P. pastoris responded to SMG. Six strongly up-regulated genes, TPX, FBA, PGAM, ENO, SBA1, and AKR-E, involved in the oxidative stress response, methanol metabolism, glycolytic pathway, and protein folding, were selected to analyze their impacts on recombinant protein production by co-overexpression in the shaker flask fermentation. The co-overexpressed strains, particularly TPX, FBA, and PGAM, demonstrated promising results with approximately 2.46-fold, 1.58-fold, and 1.33-fold increases in the specific yields of PGUS compared to the control after 48 h of methanol induction, respectively. In the meantime, the corresponding PGUS specific activities were increased by 2.33-fold, 2.09-fold, and 1.32-fold, respectively. Thiol peroxidase (TPX), which is involved in the oxidative stress response, significantly influenced the transcriptional levels of the reporter gene PGUS. The present study provides valuable information for further exploration of the molecular mechanism of P. pastoris response to SMG and facilitates simulated microgravity for finding novel helper factors to rationally engineer the strains in normal fermentation by using proteomic studies.

The detailed characterization of site-specific glycosylation requires the identification of glycan composition and specific attachment sites on proteins, which need the identification of intact glycopeptides by mass spectrometry. In this study, we present an analytical and computational strategy for the high throughput characterization of intact N-glycopeptides derived from complex proteome samples. N-glycopeptides were identified using the spectra acquired for intact glycopeptides as well as de-glycopeptides. The Y1 ion (peptide + GlcNAc) was accurately determined from the spectra of intact glycopeptides, and the structure of glycan was then identified by searching a constructed glycan database with calculated molecular weight of glycans and their fragment ions. The peptide sequences of intact glycopeptides were identified by matching the molecular weight calculated from Y1 ion to that of deglycosylated peptides from the same HILIC enrichment and identified by a separated LC–MS/MS analysis. The fully automated software platform integrates all of the above processes involved in the identification of the intact N-glycopeptides. This platform was applied to detailed characterization of site-specific glycosylation in HEK 293T cells, which led to the identification of 2249 unique intact N-glycopeptides. These intact glycopeptides revealed 1769 site-specific N-glycans on 453 glycosylation sites which demonstrated the high heterogeneity of glycosylations. Biological significance We presented a fully automated software platform for the high throughput characterization of intact N-glycopeptides derived from complex proteome samples. Intact glycopeptides and their deglycosylated forms were identified respectively and combined according to the commonality of molecular weights of peptide backbones. The strong correlation of retention times effectively filtered out random matches. The reliability of this strategy was carefully evaluated which showed a probability of random matches less than 1%. In total, 2249 intact glycopeptides were identified which is by the far the largest dataset among the studies of N-glycoproteomics.

In theory, proteases with broad cleavage specificity could be applied to digest protein samples to improve the phosphoproteomic analysis coverage. However, in practice this approach is seldom employed. This is because the identification of phosphopeptides without enzyme specificity by conventional database search strategy is extremely difficult due to the huge search space. In this study, we investigated the performance of a de novo sequencing assisted database search strategy for the identification of such phosphopeptides. Firstly, we compared the performance of conventional database search strategy and the de novo sequencing assisted database search strategy for the identification of peptides and phosphopeptides without stetting enzyme specificity. It was found that the identification sensitivity dropped significantly for the conventional one while it was only slightly decreased for the new approach. Then, this new search strategy was applied to identify phosphopeptides generated by Proteinase K digestion, which resulted in the identification of 717 phosphopeptides. Finally, this strategy was utilized for the identification of serum endogenous phosphopeptides, which were generated in vivo by different kinds of proteases and kinases, and the identification of 68 unique serum endogenous phosphopepitdes was successfully achieved.

Novel cyclodextrin (CD) chiral stationary phases (CD-CSPs) with well-defined structure have been successfully synthesized by immobilization of mono/di(10-undecenoyl)-perphenylaminocarbonyl -CD on the 3-mercaptopropyl functionalized silica gel via thiol-ene click chemistry. The phenyl carbamate groups on the rims of CD extended the cavity of CD-CSPs, which facilitated the formation of inclusion complex with various types of racemic compounds under RP mode, and also improved the - stacking interaction, dipole-dipole interaction, and hydrogen bonding interaction with racemic compounds under normal phase mode. Fifteen racemic compounds were successfully separated on this CD-CSP with HPLC, and the chromatographic results also demonstrated that thiol-ene click chemistry affords a facile approach for preparation of CSPs.

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.