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Rationale: Epithelial-mesenchymal transition (EMT) has been recognized as an important step toward high invasion and metastasis of many cancers including hepatocellular carcinoma (HCC), while the mechanism for EMT promotion is still ambiguous. Methods: The dynamic alterations of site-specific glycosylation during HGF/TGF-β1-induced EMT process of three HCC cell lines were systematically investigated using precision glycoproteomic methods. The possible roles of EMT-related glycoproteins and site-specific glycans were further confirmed by various molecular biological approaches. Results: Using mass spectrometry-based glycoproteomic methods, we totally identified 2306 unique intact glycopeptides from SMMC-7721 and HepG2 cell lines, and found that core-fucosylated glycans were accounted for the largest proportion of complex N-glycans. Through quantification analysis of intact glycopeptides, we found that the majority of core-fucosylated intact glycopeptides from folate receptor α (FOLR1) were up-regulated in the three HGF-treated cell lines. Similarly, core-fucosylation of FOLR1 were up-regulated in SMMC-7721 and Hep3B cells with TGF-β1 treatment. Using molecular approaches, we further demonstrated that FUT8 was a driver for HGF/TGF-β1-induced EMT. The silencing of FUT8 reduced core-fucosylation and partially blocked the progress of HGF-induced EMT. Finally, we confirmed that the level of core-fucosylation on FOLR1 especially at the glycosite Asn-201 positively regulated the cellular uptake capacity of folates, and enhanced uptake of folates could promote the EMT of HCC cells. Conclusions: Based on the results, we proposed a potential pathway for HGF or TGF-β1-induced EMT of HCC cells: HGF or TGF-β1 treatment of HCC cells can increase the expression of glycosyltransferase FUT8 to up-regulate the core-fucosylation of N-glycans on glycoproteins including the FOLR1; core-fucosylation on FOLR1 can then enhance the folate uptake capacity to finally promote the EMT progress of HCC cells.

Inter-tumor heterogeneity is a result of genomic, transcriptional, translational, and post-translational molecular features. To investigate the roles of protein glycosylation in the heterogeneity of high-grade serous ovarian carcinoma (HGSC), we perform mass spectrometry-based glycoproteomic characterization of 119 TCGA HGSC tissues. Cluster analysis of intact glycoproteomic profiles delineates 3 major tumor clusters and 5 groups of intact glycopeptides. It also shows a strong relationship between N-glycan structures and tumor molecular subtypes, one example of which being the association of fucosylation with mesenchymal subtype. Further survival analysis reveals that intact glycopeptide signatures of mesenchymal subtype are associated with a poor clinical outcome of HGSC. In addition, we study the expression of mRNAs, proteins, glycosites, and intact glycopeptides, as well as the expression levels of glycosylation enzymes involved in glycoprotein biosynthesis pathways in each tumor. The results show that glycoprotein levels are mainly controlled by the expression of their individual proteins, and, furthermore, that the glycoprotein-modifying glycans correspond to the protein levels of glycosylation enzymes. The variation in glycan types further shows coordination to the tumor heterogeneity. Deeper understanding of the glycosylation process and glycosylation production in different subtypes of HGSC may provide important clues for precision medicine and tumor-targeted therapy., Altered protein glycosylation is increasingly recognized as a hallmark of cancer. Here, the authors profile the glycoproteome of 119 high-grade serous ovarian carcinoma tissues, showing that glycosylation patterns correlate with tumor molecular subtypes and clinical outcomes.

International audience; The amount of protein thiols play a crucial role in maintaining the cellular redox homeostasis and have significant implications to indicate a series of diseases. Therefore, it is necessary to develop an ideal probe for protein thiol detection in a simple and readily implementable method. Consequently, a water-soluble and incubate-free fluorescent environment-sensitive probe DMTs-OCC was synthesized using 7-diethylamincoumarin as the fluorophore and 4-(5-Methanesulfonyl- [1,2,3,4]tetrazol-1-yl)-phenol (MSTP) as a thiol receptor reagent. The blue-shift emission spectra of probe DMTs-OCC was observed by ultrafast binding to protein sulfhydryl groups from the excited intramolecular charge transfer (ICT) to the twisted intramolecular charge transfer (TICT) conversion process in aqueous solution. The experimental results showed that probe DMTs-OCC exhibited an excellent selectivity to protein thiols and biocompatibility in aqueous solution, as well as terrific cell membrane permeability which enabled the successful visualization of BSA protein thiol in living cells. Moreover, no excess probe was cleaned and no incubation time was needed in cell experiments. Therefore, it could provide a new method to the construction of fluorescent probes for protein thiols labelling and visualization.

Myocardial infarction (MI) is one of the leading causes of deaths worldwide. Because of the incapability of regeneration, the cardiomyocyte loss with MI is replaced by fibrotic scar tissue, which eventually leads to heart failure. Reconstructing regeneration of an adult human heart has been recognized as a promising strategy for cardiac therapeutics. A neonatal mouse heart, which possesses transient regenerative capacity at the first week after birth, represents an ideal model to investigate processes associated with cardiac regeneration. In this work, an integrated glycoproteomic and proteomic analysis was performed to investigate the differences in glycoprotein abundances and site-specific glycosylation between postneonatal day 1 (P1) and day 7 (P7) of mouse hearts. By large-scale profiling and quantifying more than 2900 intact N-glycopeptides in neonatal mouse hearts, we identified 227 altered N-glycopeptides between P1 and P7 hearts. By extracting protein changes from the global proteome data, the normalized glycosylation changes for site-specific glycans were obtained, which showed heterogeneity on glycosites and glycoproteins. Systematic analysis of the glycosylation changes demonstrated an overall upregulation of sialylation and core fucosylation in P7 mice. Notably, the upregulated sialylation was a comprehensive result of increased sialylated glycans with Neu5Gc, with both Neu5Gc and core fucose, and decreased sialylated glycans with Neu5Ac. The upregulated core fucosylation resulted from the increase of glycans containing both core fucose and Neu5Gc but not glycans containing sole core fucose. These data provide a valuable resource for future functional and mechanism studies on heart regeneration and discovery of novel therapeutic targets. All mass spectrometry proteomic data have been deposited to the ProteomeXchange Consortium via the PRIDE partner repository with the data set identifier PXD017139.

Primary liver cancer, mainly comprising hepatocellular carcinoma (HCC) and intrahepatic cholangiocarcinoma (ICC), remains a major global health problem. Although ICC is clinically different from HCC, their molecular differences are still largely unclear. In this study, precision N-glycoproteomic analysis was performed on both ICC and HCC tumors as well as paracancer tissues to investigate their aberrant site-specific N-glycosylation. By using our newly developed glycoproteomic methods and novel algorithm, termed 'StrucGP', a total of 486 N-glycan structures attached on 1235 glycosites were identified from 894 glycoproteins in ICC and HCC tumors. Notably, glycans with uncommon LacdiNAc (GalNAcβ1-4GlcNAc) structures were distinguished from their isomeric glycans. In addition to several bi-antennary and/or bisecting glycans that were commonly elevated in ICC and HCC, a number of LacdiNAc-containing, tri-antennary, and core-fucosylated glycans were uniquely increased in ICC. More interestingly, almost all LacdiNAc-containing N-glycopeptides were enhanced in ICC tumor but not in HCC tumor, and this phenomenon was further confirmed by lectin histochemistry and the high expression of β1-4 GalNAc transferases in ICC at both mRNA and protein expression levels. The novel N-glycan alterations uniquely detected in ICC provide a valuable resource for future studies regarding to the discovery of ICC diagnostic biomarkers, therapeutic targets, and mechanism investigations.

Sialylation is involved in receptor-ligand interactions, communication between cells, and host-pathogen interactions and it is involved in the ability of glycoproteins of bovine milk to inhibit the influenza A virus (IAV). The present paper describes a simple and efficient method to isolate sialoglycoproteins from bovine milk using serotonin-magnetic particle conjugates. Then, the isolated glycoproteins were analysed by lectin blotting and LC-MS/MS. The N-glycans on isolated glycoproteins were characterized by MALDI-TOF/TOF-MS. The role of the isolated sialoglycoproteins against IAV was validated in vitro. As a result, there were 91 proteins and 17 sialylated N-glycans to be identified. The isolated proteins have ability to inhibit attachment of IAV mimics to MDCK cells. However, the role of inhibition was abolished when the sialic acid moieties were destroyed. This method could provide useful information for the large-scale production of sialoglycoproteins from bovine milk against IAV.

N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. Over the last decade, large-scale profiling of N-linked glycoproteins and glycosylation sites from biological and clinical samples has been achieved through mass spectrometry-based glycoproteomic approaches. In this paper, we reviewed the human glycoproteomic profiles that have been reported in more than 80 individual studies, and mainly focused on the N-glycoproteins and glycosylation sites identified through their deglycosylated forms of glycosite-containing peptides. According to our analyses, more than 30,000 glycosite-containing peptides and 7,000 human glycoproteins have been identified from five different body fluids, twelve human tissues (or related cell lines), and four special cell types. As the glycoproteomic data is still missing for many organs and tissues, a systematical glycoproteomic analysis of various human tissues and body fluids using a uniform platform is still needed for an integrated map of human N-glycoproteomes.

Glycosylation inhibition can improve the efficacy of antitumor drugs and enhance the apoptosis of cancer cells, thus holding great potential for cancer treatment. Inhibition of N-glycosylation induces endoplasmic reticulum (ER) stress and the unfolded protein response (UPR), and eventually triggers ER stress-related apoptosis. Unfortunately, the detailed timeline of these cell responses and protein expression alterations related to N-glycosylation inhibition is not explicit yet, and the pathways involved in different stages of N-glycosylation inhibition still need to be characterized. In this study, the dynamic proteome alterations related to N-glycosylation inhibition were investigated by further analyzing our previously published quantitative proteomics data from tunicamycin (TM)-treated ovarian carcinoma (OVCAR-3) cells. The results revealed that N-glycosylation inhibition not only directly affects the expression of glycosylated proteins but also alters an extended scale of proteins. Functional annotation of these altered proteins demonstrated that proteins related to ER stress start changing within 6 h, followed by UPR within 24 h, and eventually ER stress-related apoptosis is triggered after 48 h, indicating the conversion of cellular response from positive to negative. The dynamic proteome data presented here provide important information for better understanding of the significance of N-glycosylation to cell survival and TM-related cancer treatment.

Macrophages can be polarized into classically activated macrophages (M1) and alternatively activated macrophages (M2) in the immune system, performing pro-inflammatory and anti-inflammatory functions, respectively. Human THP-1 and mouse RAW264.7 cell line models have been widely used in various macrophage-associated studies, while the similarities and differences in protein expression profiles between the two macrophage models are still largely unclear. In this study, the protein expression profiles of M1 and M2 phenotypes from both THP-1 and RAW264.7 macrophages were systematically investigated using mass spectrometry-based proteomics. By quantitatively analyzing more than 5,000 proteins among different types of macrophages (M0, M1 and M2) from both cell lines, we identified a list of proteins that were uniquely up-regulated in each macrophage type and further confirmed 43 proteins that were commonly up-regulated in M1 macrophages of both cell lines. These results revealed considerable divergences of each polarization type between THP-1 and RAW264.7 macrophages. Moreover, the mRNA and protein expression of CMPK2, RSAD2, DDX58, and DHX58 were strongly up-regulated in M1 macrophages for both macrophage models. These data can serve as important resources for further studies of macrophage-associated diseases in experimental pathology using human and mouse cell line models.

Glycosylation is one of the most important post-translational modifications of proteins and plays an essential role in spermatogenesis, maturation, extracellular quality control, capacitation, sperm-egg recognition, and final fertilization. Spermatozoa are synthesized in the testes inactively with a thick glycocalyx and passed through the epididymis for further modification by glycosylation, deglycosylation, and integration to reach maturation. Subsequently, sperm capacitation and further fertilization require redistribution of glycoconjugates and dramatic glycocalyx modification of the spermatozoa surface. Furthermore, glycoproteins and glycans in seminal plasma are functional in maintaining spermatozoa structure and stability. Therefore, aberrant glycosylation may cause alteration of semen function and even infertility. Currently, mass spectrometry-based technologies have allowed large-scale profiling of glycans and glycoproteins in human semen. Quantitative analysis of semen glycosylation has also indicated many involved glycoproteome issues in male infertility and the potential biomarkers for diagnosis of male infertility in clinical. This review summarizes the role of glycosylation during spermatozoa development, the large-scale profiling of glycome and glycoproteome in human semen, as well as the association of aberrant glycosylation with infertility.

Precision mapping of glycans at structural and site-specific level is still one of the most challenging tasks in the glycobiology field. Here, we describe a modularization strategy for de novo interpretation of N-glycan structures on intact glycopeptides using tandem mass spectrometry. An algorithm named StrucGP is also developed to automate the interpretation process for large-scale analysis. By dividing an N-glycan into three modules and identifying each module using distinct patterns of Y ions or a combination of distinguishable B/Y ions, the method enables determination of detailed glycan structures on thousands of glycosites in mouse brain, which comprise four types of core structure and 17 branch structures with three glycan subtypes. Owing to the database-independent glycan mapping strategy, StrucGP also facilitates the identification of rare/new glycan structures. The approach will be greatly beneficial for in-depth structural and functional study of glycoproteins in the biomedical research. StrucGP offers a de novo glycan mapping method to determine detailed N-glycan structures at the site-specific level.

Hepatocellular carcinoma (HCC) is still one of the malignant tumors with high morbidity and mortality in China and worldwide. Although alpha-fetoprotein (AFP) as well as core fucosylated AFP-L3 have been widely used as important biomarkers for HCC diagnosis and evaluation, the AFP level shows a huge variation among HCC patient populations. In addition, the AFP level has also been proved to be associated with pathological grade, progression, and survival of HCC patients. Understanding the intrinsic heterogeneities of HCC associated with AFP levels is essential for the molecular mechanism studies of HCC with different AFP levels as well as for the potential early diagnosis and personalized treatment of HCC with AFP negative. In this study, an integrated N-glycoproteomic and proteomic analysis of low and high AFP levels of HCC tumors was performed to investigate the intrinsic heterogeneities of site-specific glycosylation associated with different AFP levels of HCC. By large-scale profiling and quantifying more than 4,700 intact N-glycopeptides from 20 HCC and 20 paired paracancer samples, we identified many commonly altered site-specific N-glycans from HCC tumors regardless of AFP levels, including decreased modifications by oligo-mannose and sialylated bi-antennary glycans, and increased modifications by bisecting glycans. By relative quantifying the intact N-glycopeptides between low and high AFP tumor groups, the great heterogeneities of site-specific N-glycans between two groups of HCC tumors were also uncovered. We found that several sialylated but not core fucosylated tri-antennary glycans were uniquely increased in low AFP level of HCC tumors, while many core fucosylated bi-antennary or hybrid glycans as well as bisecting glycans were uniquely increased in high AFP tumors. The data provide a valuable resource for future HCC studies regarding the mechanism, heterogeneities and new biomarker discovery.

Fucosylation (Fuc) of glycoproteins plays an important role in regulating protein function and has been associated with the development of several cancer types including prostate cancer (Pca). Therefore, the research of Fuc glycoproteins has attracted increasing attention recently in the analytical field. Herein, a strategy based on lectin affinity enrichments of intact glycopeptides followed by mass spectrometry has been established to evaluate the specificities of various Fuc-binding lectins for glycosite-specific Fuc analysis of nonaggressive (NAG) and aggressive (AG) Pca cell lines. The enrichment specificities of Fuc glycopeptides using lectins (LCA, PSA, AAL, LTL, UEA I, and AOL) and MAX extraction cartridges alone, or in tandem, were evaluated. Our results showed that the use of lectin enrichment significantly increased the ratio of fucosylated glycopeptides to total glycopeptides compared to MAX enrichment. Furthermore, tandem use of lectin followed by MAX increased the number of identifications of Fuc glycopeptides compared to using lectin enrichment alone. LCA, PSA, and AOL showed stronger binding capacity than AAL, LTL, and UEA I. Also, LCA and PSA bound specifically to core Fuc, whereas AOL, AAL, and UEA I showed binding to both core Fuc and branch Fuc. The optimized enrichment method with tandem enrichment of LCA followed by MAX (LCA-MAX) was then applied to examine the Fuc glycoproteomes in two NAG and two AG Pca cell lines. In total, 973 intact Fuc glycopeptides were identified and quantified from 252 Fuc proteins by using the tandem-mass-tags (TMT) labeling and nanoliquid chromatography-mass spectrometry (nanoLC-MS/MS) analysis. Further data analysis revealed that 51 Fuc glycopeptides were overexpressed more than 2-fold in AG cell lines compared to NAG cells. The analysis of protein core fucosylation has great potential for aiding our understanding of invasive activity of AG Pca and may lead to the development of diagnostic approaches for AG Pca.

Comprehensive analysis of the glycoproteome is critical due to the widespread importance of this post-translational modification to protein function, and difficult because of the tremendous complexity it exhibits. Here we compared three glycoproteomic analysis methods, a recently described chemoenzymatic glycoproteome analysis methods, N-linked glycans and glycosite containing peptides (NGAG), Solid-phase extraction of N-linked glycoproteins (SPEG), and hydrophilic interaction liquid chromatography (HILIC), for the analysis of N-linked glycosites of Chinese hamster ovarian (CHO) cells treated with 1,3,4-O-Bu3ManNAc. The NGAG protocol resulted in substantially increased glycosite identifications over both SPEG and HILIC. Interestingly, while the glycosites identified by SPEG and HILIC overlapped strongly, NGAG identified many glycosites not observed in either of the other two methods. Further, utilizing the enhanced intact glycopeptide identification afforded by the NGAG workflow, we also found that of the sugar analog 1,3,4-O-Bu3ManNAc increases sialylation of proteins secreted by CHO cells, including an ectopically expressed human proteins.

N-linked glycoprotein is a highly interesting class of proteins for clinical and biological research. The large-scale characterization of N-linked glycoproteins accomplished by mass spectrometry-based glycoproteomics has provided valuable insights into the interdependence of glycoprotein structure and protein function. However, these studies focused mainly on the analysis of specific sample type, and lack the integration of glycoproteomic data from different tissues, body fluids or cell types. In this study, we collected the human glycosite-containing peptides identified through their de-glycosylated forms by mass spectrometry from over 100 publications and unpublished datasets generated from our laboratory. A database resource termed N-GlycositeAtlas was created and further used for the distribution analyses of glycoproteins among different human cells, tissues and body fluids. Finally, a web interface of N-GlycositeAtlas was created to maximize the utility and value of the database. The N-GlycositeAtlas database contains more than 30,000 glycosite-containing peptides (representing > 14,000 N-glycosylation sites) from more than 7200 N-glycoproteins from different biological sources including human-derived tissues, body fluids and cell lines from over 100 studies. The entire human N-glycoproteome database as well as 22 sub-databases associated with individual tissues or body fluids can be downloaded from the N-GlycositeAtlas website at http://nglycositeatlas.biomarkercenter.org .

Background Ubiquitination is a post-translational modification where ubiquitin is covalently attached to lysine residues on substrate proteins to signal their degradation by the 26S proteasome or initiate other non-degradation functions such as cellular trafficking. The diversity of ubiquitin modifications can be attributed to the variable number of ubiquitin molecules attached to a lysine residue (mono- vs. poly-ubiquitin chains), the type of covalent linkages within poly-ubiquitin chains and the number of lysine residues on a substrate that are occupied by ubiquitin at any given time. The integral role ubiquitination plays in cell homeostasis is reflected by the multitude of diseases associated with impaired ubiquitin modification, rendering it the focus of extensive research initiatives and proteomic discovery studies. However, determining the functional role of distinct ubiquitin modifications directly from proteomic data remains challenging and represents a bottleneck in the process of deciphering how ubiquitination at specific substrate sites impacts cell signaling. Methods In this study SILAC coupled with LC–MS/MS is used to identify ubiquitinated proteins in SKOV3 ovarian cancer cells, with the implementation of a computational approach that measures relative ubiquitin occupancy at distinct modification sites upon 26S proteasome inhibition and uses that data to infer functional significance. Results In addition to identifying and quantifying relative ubiquitin occupancy at distinct post-translational modification sites to distinguish degradation from non-degradation signaling, this research led to the discovery of nine ubiquitination sites in the oncoprotein HER2 that have not been previously reported in ovarian cancer. Subsequently the computational approach applied in this study was utilized to infer the functional role of individual HER2 ubiquitin-modified residues. Conclusions In summary, the computational method, previously described for glycosylation analysis, was used in this study for the assessment of ubiquitin stoichiometries and applied directly to proteomic data to distinguish degradation from non-degradation ubiquitin functions.

Bisecting N-glycan represents one of the most important modifications to the N-glycan core, and it is involved in various biological processes. Despite many studies on the biological roles of bisecting N-glycans, current approaches for bisecting N-glycan analysis mainly rely on the use of the lectin PHA-E, which are of low specificity and sensitivity. Here, we describe a straightforward method for the recognition of bisecting N-glycans on intact glycopeptides using two characteristic Y ions [peptide+HexNAc3Hex1] and [peptide+HexNAc3Hex1Fuc1] in low energy fragmented MS/MS spectra under higher energy collisional dissociation (HCD) mode. The critical aspect of the method is the combination use of low energy HCD fragmentation and intact glycopeptide analysis. With samples from rat renal tissues, we determined the optimal fragmentation energies and analyzed the influence of core fucosylation on the intensity of the [peptide+HexNAc3Hex1] ion. Using the method, we identified 183 intact glycopeptides with bisect...

O-linked N-acetylglucosamine ( O-GlcNAc) is a ubiquitous post-translational modification of proteins and is essential for cell function. Quantifying the dynamics of O-GlcNAcylation in a proteome-wide level is critical for uncovering cellular mechanisms and functional roles of O-GlcNAcylation in cells. Here, we develop an isotope-coded photocleavable probe for profiling protein O-GlcNAcylation dynamics using quantitative mass spectrometry-based proteomics. This probe enables selective tagging and isotopic labeling of O-GlcNAcylated proteins in one step from complex cellular mixtures. We demonstrate the application of the probe to quantitatively profile O-GlcNAcylation sites in 293T cells upon chemical induction of O-GlcNAc levels. We further applied the probe to quantitatively analyze the stoichiometry of O-GlcNAcylation between sorafenib-sensitive and sorafenib-resistant liver cancer cells, which lays the foundation for mechanistic investigation of O-GlcNAcylation in regulating cancer chemoresistance. Thus, this probe provides a powerful tool to profile O-GlcNAcylation dynamics in cells.

The Chinese hamster ovary (CHO) cell line is a major expression system for the production of therapeutic proteins, the majority of which are glycoproteins, such as antibodies and erythropoietin (EPO). The characterization glycosylation profile of therapeutic proteins produced from engineered CHO cells and therapeutic functions, as well as side effects, are critical to understand the important roles of glycosylation. In this study, a large scale glycoproteomic workflow was established and applied to CHO-K1 cells expressing EPO. The workflow includes enrichment of intact glycopeptides from CHO-K1 cell lysate and medium using hydrophilic enrichment, fractionation of the obtained intact glycopeptides (IGPs) by basic reversed phase liquid chromatography (bRPLC), analyzing the glycopeptides using LC-MS/MS, and annotating the results by GPQuest 2.0. A total of 10 338 N-linked glycosite-containing IGPs were identified, representing 1162 unique glycosites in 530 glycoproteins, including 71 unique atypical N-linked IGPs on 18 atypical N-glycosylation sequons with an overrepresentation of the N-X-C motifs. Moreover, we compared the glycoproteins from CHO cell lysate with those from medium using the in-depth N-linked glycoproteome data. The obtained large scale glycoproteomic data from intact N-linked glycopeptides in this study is complementary to the genomic, proteomic, and N-linked glycomic data previously reported for CHO cells. Our method has the potential to monitor the production of recombinant therapeutic glycoproteins.

Most serum proteins are N-linked glycosylated, and therefore the glycoproteomic profiling of serum is essential for characterization of serum proteins. In this study, we profiled serum N-glycoproteome by our recently developed N-glycoproteomic method using solid-phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) coupled with LC-MS/MS and site-specific glycosylation analysis using GPQuest software. Our data indicated that half of identified N-glycosites were modified by at least two glycans, with a majority of them being sialylated. Specifically, 3/4 of glycosites were modified by biantennary N-glycans and 1/3 of glycosites were modified by triantennary sialylated N-glycans. In addition, two novel atypical glycosites (with N–X–V motif) were identified and validated from albumin and α-1B-glycoprotein. The widespread presence of these two glycosites among individuals was further confirmed by individual serum analyses.

Prostate cancer is the most common cancer among men in the U.S. and worldwide, and androgen-deprivation therapy remains the principal treatment for patients. Although a majority of patients initially respond to androgen-deprivation therapy, most will eventually develop castration resistance. An increased understanding of the mechanisms that underline the pathogenesis of castration resistance is therefore needed to develop novel therapeutics. LNCaP and PC3 prostate cancer cell lines are models for androgen-dependence and androgen-independence, respectively. Herein, we report the comparative analysis of these two prostate cancer cell lines using integrated global proteomics and glycoproteomics. Global proteome profiling of the cell lines using isobaric tags for relative and absolute quantitation (iTRAQ) labeling and two- dimensional (2D) liquid chromatography-tandem MS (LC-MS/MS) led to the quantification of 8063 proteins. To analyze the glycoproteins, glycosite-containing peptides were isolated from the same iTRAQ-labeled peptides from the cell lines using solid phase extraction followed by LC-MS/MS analysis. Among the 1810 unique N-linked glycosite-containing peptides from 653 identified N-glycoproteins, 176 glycoproteins were observed to be different between the two cell lines. A majority of the altered glycoproteins were also observed with changes in their global protein expression levels. However, alterations in 21 differentially expressed glycoproteins showed no change at the protein abundance level, indicating that the glycosylation site occupancy was different between the two cell lines. To determine the glycosylation heterogeneity at specific glycosylation sites, we further identified and quantified 1145 N-linked glycopeptides with attached glycans in the same iTRAQ-labeled samples. These intact glycopeptides contained 67 glycan compositions and showed increased fucosylation in PC3 cells in several of the examined glycosylation sites. The increase in fucosylation could be caused by the detected changes in enzymes belonging to the glycan biosynthesis pathways of protein fucosylation observed in our proteomic analysis. The altered protein fucosylation forms have great potential in aiding our understanding of castration resistance and may lead to the development of novel therapeutic approaches and specific detection strategies for prostate cancer.

Heterogeneity of protein glycosylation poses great challenges for analysis that is key to understand structure and function of glycoproteins. Resolving this conundrum requires efficient and specific enrichment of intact glycopeptides for identification and quantitation. To this end, hydrophilic interaction chromatography (HILIC) has been commonly used to enrich intact N- and O-linked glycopeptides. However, its effectiveness to enrich isobarically labeled glycopeptides remains unclear. Here, we studied three different enrichment methods for enrichment of N- and O-linked glycopeptides. It was found that removal of N-glycans prior to enrichment of O-linked glycopeptides by HILIC improved identification of O-linked glycopeptides by mass spectrometry. We also compared the enrichment of intact N- and O-linked glycopeptides using other chromatography methods and found that using cartridges containing materials for strong anion exchange (SAX) chromatography increased yield and identification of N- and O-linked glycopeptides. The enrichment of O-linked glycopeptides was further improved when a Retain AX cartridge (RAX) was used. In particular, isobaric tag labeled glycopeptides after C18 desalting could be readily enriched by SAX and RAX cartridges but not by HILIC to enable quantitative glycoproteomics. It is anticipated that the use of SAX and RAX cartridges will facilitate broad applications of identifications and quantitation of glycoproteins.

Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this "copy-paste" spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design.

HIV elite suppressors (ES) or controllers are individuals achieving control of viremia by their natural immunological mechanisms without highly active antiretroviral therapy (HAART). Study of the mechanisms responsible for the immunological suppression of viremia in ES may lead to the detection of individuals with ES and the effective control of HIV infection. We hypothesize that plasma glycoproteins play essential roles in the immune system of ES since plasma proteins are critical and highly relevant in anti-viral immunity and most plasma proteins are glycoproteins. To examine glycoproteins associated with ES, plasma samples from ES individuals (n=20), and from individuals on HAART (n=20), with AIDS (n=20), and no HIV infection (n=10) were analyzed by quantitative glycoproteomics. We found that a number of glycoproteins changed between ES versus HAART, AIDS and HIV- individuals. In sharp contrast, the level of plasma glycoproteins in the HAART cohort showed fewer changes compared with AIDS and HIV- individuals. These results showed that although both ES and HAART effectively suppress viremia, ES appeared to profoundly affect immunologically relevant glycoproteins in plasma as consequence of or support for anti-viral immunity. Bioinformatic analysis revealed that altered proteins in ES plasma were mainly associated with inflammation. This analysis suggests that overlapping, while distinguishable, glycoprotein profiles for inflammation and immune activation appeared to be present between ES and non-ES (HAART+AIDS) cohorts, indicating different triggers for inflammation and immune activation between natural and treatment-related viral suppression.

As one of the most important post-translational modifications, the discovery, isolation, and identification of glycoproteins are becoming increasingly important. In this study, a Con A-magnetic particle conjugate-based method was utilized to selectively isolate the glycoproteins and their glycomes from the healthy donor and hepatocellular carcinoma (HCC) case sera. The isolated glycoproteins and their N-linked glycans were identified by LC-ESI-MS/MS and MALDI-TOF/TOF-MS, respectively. A total of 93 glycoproteins from the healthy donors and 85 glycoproteins from the HCC cases were identified. There were 34 different glycoproteins shown between the healthy donors (21/34) and the HCC cases (13/34). Twenty-eight glycans from the healthy donors and 30 glycans from the HCC cases were detected and there were 22 different glycans shown between the healthy donors (10/22) and HCC cases (12/22). Among these glycoproteins, 50 were known to be N-linked glycoproteins and three novel glycopeptides from two predicted potential glycoproteins were discovered. Moreover, lectin blotting, Western blotting and lectin/glyco-antibody microarrays were applied to definitely elucidate the change of selective protein expressions and their glycosylation levels, the results indicated that the differences of the identified glycoproteins between the healthy donors and HCC cases were caused by the change of both protein expression and their glycosylation levels.

Analysis of intact glycopeptides by mass spectrometry is essential to determining the microheterogeneity of protein glycosylation. Higher-energy collisional dissociation (HCD) fragmentation of glycopeptides generates mono- or disaccharide ions called oxonium ions that carry information about the structure of the fragmented glycans. Here, we investigated the link between glycan structures and the intensity of oxonium ions in the spectra of glycopeptides and utilized this information to improve the identification of glycopeptides in biological samples. Tandem spectra of glycopeptides from fetuin, glycophorin A, ovalbumin and gp120 tryptic digests were used to build a spectral database of N- and O-linked glycopeptides. Logistic regression was applied to this database to develop model to distinguish between the spectra of N- and O-linked glycopeptides. Remarkably, the developed model was found to reliably distinguish between the N- and O-linked glycopeptides using the spectral features of the oxonium ions using verification spectral set. Finally, the performance of the developed predictive model was evaluated in HILIC enriched glycopeptides extracted from human serum. The results showed that pre-classification of tandem spectra based on their glycosylation type improved the identification of N-linked glycopeptides. The developed model facilitates interpretation of tandem mass spectrometry data for assignment of glycopeptides.

Influenza is an acute viral disease of the respiratory tract that affects millions of people each year. Glycan plays an important role in the life cycle of influenza virus. The study of N-linked glycosylation and related virus biology has become an area of intense interest in recent years due to its ability to impart various advantages to virus survival and virulence as well as host specificity. For example, the sialylated glycans on the host cell surface were the specific receptor for influenza virus and viruses use this host cell process to modify proteins present on their surface, which ultimately impacts the role of the viral glycoproteins in stability, antigenicity and host cell invasion. Here, the recent findings on the role of glycosylation in virus biology and host response to viral infection and the factors that determine the glycosylation of its surface proteins hemagglutinin (HA) are reviewed. Furthermore, the effects of host-specific glycosylation on receptor binding, fusion activity, antigenic properties of the virus and selection on virulence as well as its effects on host range are emphasized.

Influenza H1N1 virus has posed a serious threat to human health. The glycosylation of neuraminidase (NA) could affect the infectivity and virulence of the influenza virus, but detailed site-specific glycosylation information of NA is still missing. In this study, intact glycopeptide analysis is performed on an influenza NA (A/H1N1/California/2009) that is expressed in human 293T and insect Hi-5 cells. The data indicate that three of four potential N-linked glycosylation sites are glycosylated, including one partial glycosylation site from both cell lines. The NA expressed in human cells has more complex glycans than that of insect cells, suggesting the importance of selecting an appropriate expression system for the production of functional glycoproteins. Different types of glycans are identified from different glycosites of NA expressed in human cells, which implies the site-dependence of glycosylation on NA. This study provides valuable information for the research of influenza virus as well as the functions of viral protein glycosylation.

It is well-known that N-linked glycans usually attach to asparagine residues in the N-X-S/T motifs of proteins. However, accumulating evidence indicates that N-glycosylation could also possibly occur at other atypical motifs. In this study, we tried to identify atypical N-glycosylation sites using our recently developed solid-phase extraction of the N-linked Glycans And Glycosite-containing peptides (NGAG) method. Peptides with deamidation sites at asparagine residues but lacking a typical asparagine-X-serine/threonine sequons (N-X-S/T, X is any amino acid except proline) motif were identified from deglycosylated peptide data as potentially atypical glycosite-containing peptides. These atypical glycosites were verified by the presence of glycans on their intact glycopeptides and further confirmed by specific inhibition of cells with an N-linked glycosylation inhibitor, tunicamycin. From this study, two atypical N-linked glycosylation sites with N-X-C and N-X-V motifs were identified and validated from an ovarian cancer cell line (OVCAR-3).

Protein glycosylation is one of the most important protein modifications. Glycosylation site occupancy alteration has been implicated in human diseases and cancers. However, current glycoproteomic methods focus on the identification and quantification of glycosylated peptides and glycosylation sites but not glycosylation occupancy or glycoform stoichiometry. Here we describe a method for large-scale determination of the absolute glycosylation stoichiometry using three independent relative ratios. Using this method, we determined 117 absolute N-glycosylation occupancies in OVCAR-3 cells. Finally, we investigated the possible functions and the determinants for partial glycosylation.

Comprehensive characterization of protein glycosylation is critical for understanding the structure and function of glycoproteins. However, due to the complexity and heterogeneity of glycoprotein conformations, current glycoprotein analyses focus mainly on either the de-glycosylated glycosylation site (glycosite)-containing peptides or the released glycans. Here, we describe a chemoenzymatic method called solid phase extraction of N-linked glycans and glycosite-containing peptides (NGAG) for the comprehensive characterization of glycoproteins that is able to determine glycan heterogeneity for individual glycosites in addition to providing information about the total N-linked glycan, glycosite-containing peptide and glycoprotein content of complex samples. The NGAG method can also be applied to quantitatively detect glycoprotein alterations in total and site-specific glycan occupancies.

Hepatoblastoma (HB) is the most common form of liver tumour in infants and children. The cancer cell line HepG2 and normal hepatocellular cell line L02 are valuable cell models and are already widely used in the world while their glycopeoteome profiles are still unknown. This study focuses on N-linked glycoproteomic analysis of these two cell lines using mass spectrometry. Using two complementary approaches, Hydrazide reaction and hydrophilic affinity solid phase extraction methods, almost 400 glycosylation sites were identified from the two cell lines. Functional annotation suggests that N-glycoproteins with molecular binding ability were enriched in HepG2 cells compared to normal liver cells.

Glycoproteins secreted into plasma from T cells infected with human immunodeficiency virus (HIV) latent infection may provide insight into understanding the host response to HIV infection in vivo. Glycoproteomics, which evaluates the level of the glycoproteome, remains a novel approach to study this host response to HIV. In order to identify human glycoproteins secreted from T cells with latent HIV infection, the medium from cultured HIV replication-competent T cells was compared with the medium from cultured parental A3.01 cells via solid phase extraction of glycopeptides (SPEG) and high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS). Using these methods, 59 human glycoproteins were identified as having significantly different abundance levels between the media from these two cell lines. The relevance of these 59 proteins to HIV infection in vivo was assessed in plasma from HIV+ and HIV- subjects. Comparison between T cell and plasma revealed that six glycoproteins (galectin-3-binding protein, L-selectin, neogenin, adenosine deaminase CECR1, ICOS ligand and phospholipid transfer protein) were significantly elevated in the HIV+ T cells and plasma studies. These findings suggest that the response of T cells harboring latent HIV infection contributed, in part, to the glycoprotein changes in HIV+ plasma. These proteins, once validated, could provide insight into host-HIV interaction.

Protein glycosylation has long been recognized as one of the most common post-translational modifications. Most membrane proteins and extracellular proteins are N-linked glycosylated, and they account for the majority of current clinical diagnostic markers or therapeutic targets. Quantitative proteomic analysis of detectable N-linked glycoproteins from cells or tissues using mass spectrometry has the potential to provide biological basis for disease development and identify disease associated glycoproteins. However, the information of low abundance but important peptides is lost due to the lack of MS/MS fragmentation or low quality of MS/MS spectra for low abundance peptides. Here, we show the feasibility of formerly N-glycopeptide identification and quantification at MS1 level using genomic N-glycosite prediction (GenoGlyco) coupled with stable isotopic labeling and accurate mass matching. The GenoGlyco Analyzer software uses accurate precursor masses of detected N-deglycopeptide peaks to match them to N-linked deglycopeptides that are predicted from genes expressed in the cells. This method results in more robust glycopeptide identification compared to MS/MS-based identification. Our results showed that over three times the quantity of N-deglycopeptide assignments from the same mass spectrometry data could be produced in ovarian cancer cell lines compared to a MS/MS fragmentation method. Furthermore, the method was also applied to N-deglycopeptide analysis of ovarian tumors using the identified deglycopeptides from the two ovarian cell lines as heavy standards. We show that the described method has a great potential in the analysis of detectable N-glycoproteins from cells and tissues.

Urea solution is one of the most commonly employed protein denaturants for protease digestion in proteomic studies. However, it has long been recognized that urea solution can cause carbamylation at the N termini of proteins/peptides and at the side chain amino groups of lysine and arginine residues. Protein/peptide carbamylation blocks protease digestion and affects protein identification and quantification in mass spectrometry analysis by blocking peptide amino groups from isotopic/isobaric labeling and changing peptide charge states, retention times, and masses. In addition, protein carbamylation during sample preparation makes it difficult to study in vivo protein carbamylation. In this study, we compared the peptide carbamylation in urea solutions of different buffers and found that ammonium-containing buffers were the most effective buffers to inhibit protein carbamylation in urea solution. The possible mechanism of carbamylation inhibition by ammonium-containing buffers is discussed, and a revised procedure for the protease digestion of proteins in urea and ammonium-containing buffers was developed to facilitate its application in proteomic research.

Platelet glycoproteins are known to play central roles in hemostasis and vascular integrity and have pathologic roles in vascular occlusive diseases such as myocardial infarction and stroke. Characterizing glycoproteins within and secreted by platelets can provide insight into the mechanisms that underlie vascular pathologies and the therapeutic benefits or failure of anti-platelet agents. To study the impact of aspirin, which is commonly prescribed for primary and secondary cardiovascular prevention, on the platelet glycoproteome, we evaluated washed platelets from ten donors. The platelet glycoproteome, was studied using an iTRAQ in resting and stimulated states and with and without aspirin treatment. Using solid phase extraction of glycosite-containing peptides (SPEG), we were able to identify 799 unique N-linked glycosylation sites (glycosites) in platelets, representing the largest and the most comprehensive analysis to date. We were able to identity a number of glycoproteins impacted by aspirin treatment, which we validated using global proteomics analysis of platelets and their secreted proteins. In our analyses, metallopeptidase inhibitor 1 (TIMP1) was the single most significantly affected glycoprotein by aspirin treatment. ELISA assays confirmed proteomic results and validated our strategy. Functional analysis demonstrated that TIMP1 levels were highly correlated with platelet reactivity in vitro, with a correlation coefficient of -0.5. The release of TIMP1 from platelets, which was previously unknown to be affected by aspirin treatment, may play important roles in hemostasis and/or vascular integrity. If validated, our findings may be useful for developing assays that assess platelet response to aspirin or other anti-platelet therapies.

Two glycoproteins, hemagglutinin (HA) and neuraminidase (NA), on the surface of influenza viruses play crucial roles in transfaunation, membrane fusion and the release of progeny virions. To explore the distribution of N-glycosylation sites (glycosites) in these two glycoproteins, we collected and aligned the amino acid sequences of all the HA and NA subtypes. Two glycosites were located at HA0 cleavage sites and fusion peptides and were strikingly conserved in all HA subtypes, while the remaining glycosites were unique to their subtypes. Two to four conserved glycosites were found in the stalk domain of NA, but these are affected by the deletion of specific stalk domain sequences. Another highly conserved glycosite appeared at the top center of tetrameric global domain, while the others glycosites were distributed around the global domain. Here we present a detailed investigation of the distribution and the evolutionary pattern of the glycosites in the envelope glycoproteins of IVs, and further focus on the H5N1 virus and conclude that the glycosites in H5N1 have become more complicated in HA and less influential in NA in the last five years.

Protein glycosylation alteration is typically employed by various viruses for escaping immune pressures from their hosts. Our previous work had shown that not only the increase of glycosylation sites (glycosites) numbers, but also glycosite migration might be involved in the evolution of human seasonal influenza H1N1 viruses. More importantly, glycosite migration was likely a more effectively alteration way for the host adaption of human influenza H1N1 viruses. In this study, we provided more bioinformatics and statistic evidences for further predicting the significant biological functions of glycosite migration in the host adaptation of human influenza H1N1 viruses, by employing homology modeling and in silico protein glycosylation of representative HA and NA proteins as well as amino acid variability analysis at antigenic sites of HA and NA. The results showed that glycosite migrations in human influenza viruses have at least five possible functions: to more effectively mask the antigenic sites, to more effectively protect the enzymatic cleavage sites of neuraminidase (NA), to stabilize the polymeric structures, to regulate the receptor binding and catalytic activities and to balance the binding activity of hemagglutinin (HA) with the release activity of NA. The information here can provide some constructive suggestions for the function research related to protein glycosylation of influenza viruses, although these predictions still need to be supported by experimental data.

Influenza virus typically alters protein glycosylation in order to escape immune pressure from hosts and hence to facilitate survival in different host environments. In this study, the patterns and conservation of glycosylation sites on HA and NA of influenza A/H1N1 viruses isolated from various hosts at different time periods were systematically analyzed, by employing a new strategy combining genome-based glycosylation site prediction and 3D modeling of glycoprotein structures, for elucidation of the modes and laws of glycosylation site alteration in the evolution of influenza A/H1N1 viruses. The results showed that influenza H1N1 viruses underwent different alterations of protein glycosylation in different hosts. Two alternative modes of glycosylation site alteration were involved in the evolution of human influenza virus: One was an increase in glycosylation site numbers, which mainly occurred with high frequency in the early stages of evolution. The other was a change in the positional conversion of the glycosylation sites, which was the dominating mode with relatively low frequency in the later evolutionary stages. The mechanisms and possibly biological functions of glycosylation site alteration for the evolution of influenza A/H1N1 viruses were also discussed. Importantly, the significant role of positional alteration of glycosylation sites in the host adaptation of influenza virus was elucidated. Although the results still need to be supported by experimental data, the information here may provide some constructive suggestions for research into the glycosylation of influenza viruses as well as even the design of surveillance and the production of viral vaccines.

Glycan-protein interactions play important biological roles in biological processes. Although there are some methods such as glycan arrays that may elucidate recognition events between carbohydrates and protein as well as screen the important glycan-binding proteins, there is a lack of simple effectively separate method to purify them from complex samples. In proteomics studies, fractionation of samples can help to reduce their complexity and to enrich specific classes of proteins for subsequent downstream analyses. Herein, a rapid simple method for purification of glycan-binding proteins from proteomic samples was developed using hydroxyl-coated magnetic particles coupled with underivatized carbohydrate. Firstly, the epoxy-coated magnetic particles were further hydroxyl functionalized with 4-hydroxybenzhydrazide, then the carbohydrates were efficiently immobilized on hydroxyl functionalized surface of magnetic particles by formation of glycosidic bond with the hemiacetal group at the reducing end of the suitable carbohydrates via condensation. All conditions of this method were optimized. The magnetic particle-carbohydrate conjugates were used to purify the glycan-binding proteins from human serum. The fractionated glycan-binding protein population was displayed by SDS-PAGE. The result showed that the amount of 1 mg magnetic particles coupled with mannose in acetate buffer (pH 5.4) was 10 micromol. The fractionated glycan-binding protein population in human serum could be eluted from the magnetic particle-mannose conjugates by 0.1% SDS. The methodology could work together with the glycan microarrays for screening and purification of the important GBPs from complex protein samples.

Increasing numbers of H5N1 influenza viruses (IVs) are responsible for human deaths, especially in North Africa and Southeast Asian. The binding of hemagglutinin (HA) on the viral surface to host sialic acid (SA) receptors is a requisite step in the infection process. Phylogenetic analysis reveals that H5N1 viruses can be divided into 10 clades based on their HA sequences, with most human IVs centered from clade 1 and clade 2.1 to clade 2.3. Protein sequence alignment in various clades indicates the high conservation in the receptor-binding domains (RBDs) is essential for binding with the SA receptor. Two glycosylation sites, 158N and 169N, also participate in receptor recognition. In the present work, we attempted to construct a serial H5N1 HA models including diverse glycosylated HAs to simulate the binding process with various SA receptors in silico. As the SA-α-2,3-Gal and SA-α-2,6-Gal receptor adopted two distinctive topologies, straight and fishhook-like, respectively, the presence of N-glycans at 158N would decrease the affinity of HA for all of the receptors, particularly SA-α-2,6-Gal analogs. The steric clashes of the huge glycans shown at another glycosylation site, 169N, located on an adjacent HA monomer, would be more effective in preventing the binding of SA-α-2,3-Gal analogs.