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1.  [No title available] 
The fact that most proteins are glycosylated underlies the key roles played by glycosylation during evolution. The functions of glycoproteins are tremendously broad and include cell attachment recognition, homeostasis, transport of molecules, and enzymatic and immunology recognition domains. One of the principal axes of glycoprotein research is to understand better the correlation between glycan structure and function. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information.
The goal of the gPRG 2013 study was to determine the ability of the international glycoproteomics community to compare N-glycosylation between two different sources of prostate specific antigen (PSA) by mass spectrometry. PSA was selected to conduct this study for several reasons: PSA is a relatively small glycoprotein (MW ∼30,000Da) characterized by a single site of N-glycosylation and is of significant biological interest as a biomarker for prostate cancer.
A total of 35 samples were sent to laboratories around the world, of which 25 data sets were returned. Here, we present the study results, and a global overview of the approaches and methodologies used for differential glycoprotein characterization. The results highlight the challenges faced by researchers in this glycoprotein characterization. We will present a consensus of the interlaboratory data collection built using statistical treatment so as to compare qualitatively and quantitatively the data submitted. In this presentation we will answer the following questions: Which sample preparation, separation and analysis methods produced the most consistent results?What is the consensus for the glycosylation patterns of the two sources of PSA?For which glycoforms from the two PSA sources are abundances significantly different?
PMCID: PMC3635324
2.  Glycoprotein Research Group (gPRG) 2013 Interlaboratory Study: Quantitative N-glycan Profiling of Prostate Specific Antigen 
The fact that most proteins are glycosylated underlies the key roles played by glycosylation during evolution. The functions of glycoproteins are tremendously broad and include cell attachment recognition, homeostasis, transport of molecules, and enzymatic and immunology recognition domains. One of the principal axes of glycoprotein research is to understand better the correlation between glycan structure and function. Mass spectrometry has emerged as a powerful analytical technique in the field of glycoprotein characterization. Its sensitivity, high dynamic range, and mass accuracy provide both quantitative and sequence/structural information.
The goal of the gPRG 2013 study was to determine the ability of the international glycoproteomics community to compare N-glycosylation between two different sources of prostate specific antigen (PSA) by mass spectrometry. PSA was selected to conduct this study for several reasons: PSA is a relatively small glycoprotein (MW ∼30,000Da) characterized by a single site of N-glycosylation and is of significant biological interest as a biomarker for prostate cancer.
A total of 35 samples were sent to laboratories around the world, of which 25 data sets were returned. Here, we present the study results, and a global overview of the approaches and methodologies used for differential glycoprotein characterization. The results highlight the challenges faced by researchers in this glycoprotein characterization. We will present a consensus of the interlaboratory data collection built using statistical treatment so as to compare qualitatively and quantitatively the data submitted. In this presentation we will answer the following questions: Which sample preparation, separation and analysis methods produced the most consistent results?What is the consensus for the glycosylation patterns of the two sources of PSA?For which glycoforms from the two PSA sources are abundances significantly different?
PMCID: PMC3635456
3.  Glycoproteomic and glycomic databases 
Clinical proteomics  2014;11(1):15.
Protein glycosylation serves critical roles in the cellular and biological processes of many organisms. Aberrant glycosylation has been associated with many illnesses such as hereditary and chronic diseases like cancer, cardiovascular diseases, neurological disorders, and immunological disorders. Emerging mass spectrometry (MS) technologies that enable the high-throughput identification of glycoproteins and glycans have accelerated the analysis and made possible the creation of dynamic and expanding databases. Although glycosylation-related databases have been established by many laboratories and institutions, they are not yet widely known in the community. Our study reviews 15 different publicly available databases and identifies their key elements so that users can identify the most applicable platform for their analytical needs. These databases include biological information on the experimentally identified glycans and glycopeptides from various cells and organisms such as human, rat, mouse, fly and zebrafish. The features of these databases - 7 for glycoproteomic data, 6 for glycomic data, and 2 for glycan binding proteins are summarized including the enrichment techniques that are used for glycoproteome and glycan identification. Furthermore databases such as Unipep, GlycoFly, GlycoFish recently established by our group are introduced. The unique features of each database, such as the analytical methods used and bioinformatical tools available are summarized. This information will be a valuable resource for the glycobiology community as it presents the analytical methods and glycosylation related databases together in one compendium. It will also represent a step towards the desired long term goal of integrating the different databases of glycosylation in order to characterize and categorize glycoproteins and glycans better for biomedical research.
doi:10.1186/1559-0275-11-15
PMCID: PMC3996109  PMID: 24725457
4.  Nanotechnologies in Glycoproteomics 
Clinical proteomics  2014;11(1):21.
Protein glycosylation, as an important post-translational modification, is implicated in a number of ailments. Applying proteomic approaches, including mass spectrometry (MS) analyses that have played a significant role in biomarker detection and early diagnosis of diseases, to the study of glycoproteins or glycopeptides will facilitate a deeper understanding of many physiological functions and biological pathways involved in cancer, inflammatory and degenerative diseases. The abundance of glycopeptides and their ionization potential are relatively lower compared to those of non-glycopeptides; therefore, sample enrichment is necessary for glycopeptides prior to MS analysis. The application of nanotechnology in the past decade has been rapidly penetrating into many diverse scientific research disciplines. Particularly in what we now refer to as the “glycoproteomics area”, nanotechnologies have enabled enhanced sensitivity and specificity of glycopeptide detection in complex biological fluids, which are critical for disease diagnosis and monitoring. In this review, we highlight some recent studies that combine the capabilities of specific nanotechnologies with the comprehensive features of glycoproteomics. In particular, we focus on the ways in which nanotechnology has facilitated the detection of glycopeptides in complex biological samples and enhanced their characterization by MS, in terms of intensity and resolution. These studies reveal an increasingly important role for nanotechnology in helping to overcome certain technical challenges in biomarker discovery, in general, and glycoproteomics research, in particular.
doi:10.1186/1559-0275-11-21
PMCID: PMC4040410  PMID: 24940182
5.  Characterization of the membrane proteome and N-glycoproteome in BV-2 mouse microglia by liquid chromatography-tandem mass spectrometry 
BMC Genomics  2014;15:95.
Background
Microglial cells are resident macrophages of the central nervous system and important cellular mediators of the immune response and neuroinflammatory processes. In particular, microglial activation and communication between microglia, astrocytes, and neurons are hallmarks of the pathogenesis of several neurodegenerative diseases. Membrane proteins and their N-linked glycosylation mediate this microglial activation and regulate many biological process including signal transduction, cell-cell communication, and the immune response. Although membrane proteins and N-glycosylation represent a valuable source of drug target and biomarker discovery, the knowledge of their expressed proteome in microglia is very limited.
Results
To generate a large-scale repository, we constructed a membrane proteome and N-glycoproteome from BV-2 mouse microglia using a novel integrated approach, comprising of crude membrane fractionation, multienzyme-digestion FASP, N-glyco-FASP, and various mass spectrometry. We identified 6928 proteins including 2850 membrane proteins and 1450 distinct N-glycosylation sites on 760 N-glycoproteins, of which 556 were considered novel N-glycosylation sites. Especially, a total of 114 CD antigens are identified via MS-based analysis in normal conditions of microglia for the first time. Our bioinformatics analysis provides a rich proteomic resource for examining microglial function in, for example, cell-to-cell communication and immune responses.
Conclusions
Herein, we introduce a novel integrated proteomic approach for improved identification of membrane protein and N-glycosylation sites. To our knowledge, this workflow helped us to obtain the first and the largest membrane proteomic and N-glycoproteomic datesets for mouse microglia. Collectively, our proteomics and bioinformatics analysis significantly expands the knowledge of the membrane proteome and N-glycoproteome expressed in microglia within the brain and constitutes a foundation for ongoing proteomic studies and drug development for various neurological diseases.
doi:10.1186/1471-2164-15-95
PMCID: PMC3938046  PMID: 24495382
Microglia; Membrane proteome; N-glycoproteome; Proteomics; Crude membrane fractionation; FASP; N-glyco-FASP
6.  Evaluation of Two Complementary Methods for Quantitative Profiling of PSA N-Glycans and N-Glycopeptides 
The analytical methods for the structural characterization of protein glycosylation in glycomics and glycoproteomics are gradually becoming more suitable for biological applications. Over the past 10 years there has been a concerted effort to increase the sensitivity, speed and automation of the analysis of more complex glycoprotein mixtures at the level of biologically relevant dynamic ranges. However, these promising analytical tools, which predominantly are mass spectrometry based, needs thorough comparison and validation, in particular when quantitation is required. As a part of the ABRF Glycoprotein Research Group (gPRG) Quantitative Glycoprotein Study, we have profiled the single N-glycosylation of two sources of human prostate specific antigen (PSA) using two different, but commonly used, analytical methods allowing us to compare their qualitative and quantitative performance: i) Quantitative site-specific analysis of enriched PSA N-glycopeptides and ii) quantitative global analysis of released and reduced PSA N-glycans. For both approaches porous graphitized carbon LC connected directly with ion trap MS/MS (CID) was used for analyte separation and detection, respectively, but with different MS polarity mode detection. Using label-free relative quantitation, the two analytical methods produced very similar PSA glycoprofiles considering their different nature. Of the 40-50 monosaccharide compositions detected from each PSA variant, mostly minor quantitative differences in the glycoprofiles produced by the two approaches were observed, enhancing the confidence of the analysis. The pros and cons of the two glycoprofiling approaches and the intended near-future improvements will be discussed.
PMCID: PMC3635316
7.  Analytical Performance of Immobilized Pronase for Glycopeptide Footprinting and Implications for Surpassing Reductionist Glycoproteomics 
Journal of proteome research  2009;8(2):502-512.
A fully developed understanding of protein glycosylation requires characterization of the modifying oligosaccharides, elucidation of their covalent attachment sites, and determination of the glycan heterogeneity at specific sites. Considering the complexity inherent to protein glycosylation, establishing these features for even a single protein can present an imposing challenge. In order to meet the demands of glycoproteomics, the capability to screen far more complex systems of glycosylated proteins must be developed. Although the proteome wide examination of carbohydrate modification has become an area of keen interest, the intricacy of protein glycosylation has frustrated the progress of large scale, systems oriented research on site-specific protein-glycan relationships. Indeed, the analytical obstacles in this area have been more instrumental in shaping the current glycoproteomic paradigm than have the diverse functional roles and ubiquitous nature of glycans. This report describes the ongoing development and analytically salient features of bead immobilized pronase for glycosylation site footprinting. The present work bears on the ultimate goal of providing analytical tools capable of addressing the diversity of protein glycosylation in a more comprehensive and efficient manner. In particular, this approach has been assessed with respect to reproducibility, sensitivity, and tolerance to sample complexity. The efficiency of pronase immobilization, attainable pronase loading density, and the corresponding effects on glycoprotein digestion rate were also evaluated. In addition to being highly reproducible, the immobilized enzymes retained a high degree of proteolytic activity after repeat usage for up to 6 weeks. This method also afforded a low level of chemical background and provided favorable levels of sensitivity with respect to traditional glycoproteomic strategies. Thus, the application of immobilized pronase shows potential to contribute to the advancement of more comprehensive glycoproteomic research methods that are capable of providing site-specific glycosylation and microheterogeneity information across many proteins.
doi:10.1021/pr800708h
PMCID: PMC2637306  PMID: 19072223
Enzyme immobilization; glycopeptide footprinting; glycoproteomics; pronase; protein glycosylation; site-specific glycosylation analysis
8.  Recent Advances in the Mass Spectrometric Analysis of Glycoproteins: Theoretical Considerations 
Electrophoresis  2010;32(1):3-13.
Protein glycosylation is involved in a broad range of biological processes that regulate protein function and control cell fate. As aberrant glycosylation has been found to be implicated in numerous diseases, the study and large-scale characterization of protein glycosylation is of great interest not only to the biological and biomedical research community, but also to the pharmaceutical and biotechnology industry. Due to the complex chemical structure and differing chemical properties of the protein/peptide and glycan moieties, the analysis and structural characterization of glycoproteins has been proven to be a difficult task. Large-scale endeavors have been further limited by the dynamic outcome of the glycosylation process itself, and, occasionally, by the low abundance of glycoproteins in biological samples. Recent advances in mass spectrometry (MS) instrumentation, and progress in miniaturized technologies for sample handling, enrichment and separation, have resulted in robust and compelling analysis strategies that effectively address the challenges of the glycoproteome. This review summarizes the key steps that are involved in the development of efficient glycoproteomic analysis methods, and the latest innovations that led to successful strategies for the characterization of glycoproteins and their corresponding glycans. As a follow-up to this work, we review innovative capillary and microfluidic-MS workflows for the identification, sequencing, and characterization of glycoconjugates.
doi:10.1002/elps.201000393
PMCID: PMC3717296  PMID: 21171109
9.  Differential analysis of N-glycoproteome between hepatocellular carcinoma and normal human liver tissues by combination of multiple protease digestion and solid phase based labeling 
Clinical Proteomics  2014;11(1):26.
Background
Dysregulation of glycoproteins is closely related with many diseases. Quantitative proteomics methods are powerful tools for the detection of glycoprotein alterations. However, in almost all quantitative glycoproteomics studies, trypsin is used as the only protease to digest proteins. This conventional method is unable to quantify N-glycosites in very short or long tryptic peptides and so comprehensive glycoproteomics analysis cannot be achieved.
Methods
In this study, a comprehensive analysis of the difference of N-glycoproteome between hepatocellular carcinoma (HCC) and normal human liver tissues was performed by an integrated workflow combining the multiple protease digestion and solid phase based labeling. The quantified N-glycoproteins were analyzed by GoMiner to obtain a comparative view of cellular component, biological process and molecular function.
Results/conclusions
An integrated workflow was developed which enabled the processes of glycoprotein coupling, protease digestion and stable isotope labeling to be performed in one reaction vessel. This workflow was firstly evaluated by analyzing two aliquots of the same protein extract from normal human liver tissue. It was demonstrated that the multiple protease digestion improved the glycoproteome coverage and the quantification accuracy. This workflow was further applied to the differential analysis of N-glycoproteome of normal human liver tissue and that with hepatocellular carcinoma. A total of 2,329 N-glycosites on 1,052 N-glycoproteins were quantified. Among them, 858 N-glycosites were quantified from more than one digestion strategy with over 99% confidence and 1,104 N-glycosites were quantified from only one digestion strategy with over 95% confidence. By comparing the GoMiner results of the N-glycoproteins with and without significant changes, the percentage of membrane and secreted proteins and their featured biological processes were found to be significant different revealing that protein glycosylation may play the vital role in the development of HCC.
doi:10.1186/1559-0275-11-26
PMCID: PMC4112855  PMID: 25097464
N-glycoproteome; N-glycosite; Multiple protease digestion; Quantitative analysis
10.  Targeting the glycoproteome 
Glycoconjugate Journal  2012;30(2):119-136.
Despite numerous original publications describing the structural complexity of N- and O-linked glycans on glycoproteins, only very few answer the basic question of which particular glycans are linked to which amino acid residues along the polypeptide chain. Such structural information is of fundamental importance for understanding the biological roles of complex glycosylations as well as deciphering their non-template driven biosynthesis. This review focuses on presenting and commenting on recent strategies, specifically aimed at identifying the glycoproteome of cultured cells and biological samples, using targeted and global enrichment procedures and utilizing the high resolution power, high through-put capacity and complementary fragmentation techniques of tandem mass spectrometry. The goal is to give an update of this emerging field of protein and glyco-sciences and suggest routes to bridge the data gap between the two aspects of glycoprotein characteristics, i.e. glycan structures and their attachment sites.
doi:10.1007/s10719-012-9438-6
PMCID: PMC3552370  PMID: 22886069
Glycoproteomics; Glycopeptide; Attachment sites; Liquid chromatography; Tandem mass spectrometry; Enrichment; Lectin affinity; Hydrazide chemistry
11.  Computational analysis of Concanavalin A binding glycoproteins of human seminal plasma 
Bioinformation  2011;7(2):69-75.
Glycoproteins have immense clinical importance and comparative glycoproteomics has become a powerful tool for biomarker discovery and disease diagnosis. Seminal plasma glycoproteins participate in fertility related processes including sperm-egg recognition, modulation of capacitation and acrosome reaction inhibition. Affinity chromatography using broad specificity lectin such as Con A is widely applied for glycoproteins enrichment. More notably, Con A-interacting fraction of human seminal plasma has decapacitating activity which makes this fraction critically important. In our previous study, we isolated Con A-interacting glycoproteins from human seminal plasma and subsequently identified them by mass spectrometry. Here, we report the computational analysis of these proteins using bioinformatics tools. The analysis includes: prediction of glycosylation sites using sequence information (NetNGlyc 1.0), functional annotations to cluster these proteins into various functional groups (InterProScan and Blast2GO) and identification of protein interaction networks (STRING database). The results indicate that these proteins are involved in various biological processes including transport, morphogenesis, metabolic processes, cell differentiation and homeostasis. The clusters illustrate two major molecular functions - hydrolase activity (6) and protein (4)/carbohydrate (1)/lipid binding (1). The large interactomes of proteins point towards their versatile roles in wide range of biological processes.
PMCID: PMC3174039  PMID: 21938208
12.  Glycoproteomics in Neurodegenerative Diseases 
Mass spectrometry reviews  2010;29(1):79-125.
Protein glycosylation regulates protein function and cellular distribution. Additionally, aberrant protein glycosylations have been recognized to play major roles in human disorders, including neurodegenerative diseases. Glycoproteomics, a branch of proteomics that catalogs and quantifies glycoproteins, provides a powerful means to systematically profile the glycopeptides or glycoproteins of a complex mixture that are highly enriched in body fluids, and therefore, carry great potential to be diagnostic and/or prognostic markers. Application of this mass spectrometry-based technology to the study of neurodegenerative disorders (e.g., Alzheimer's disease and Parkinson's disease) is relatively new, and is expected to provide insight into the biochemical pathogenesis of neurodegeneration, as well as biomarker discovery. In this review, we have summarized the current understanding of glycoproteins in biology and neurodegenerative disease, and have discussed existing proteomic technologies that are utilized to characterize glycoproteins. Some of the ongoing studies, where glycoproteins isolated from cerebrospinal fluid and human brain are being characterized in Parkinson's disease at different stages versus controls, are presented, along with future applications of targeted validation of brain specific glycoproteins in body fluids.
doi:10.1002/mas.20221
PMCID: PMC2799547  PMID: 19358229
glycoproteomics; mass spectrometry; Alzheimer's diseases; Parkinson's disease; biomarkers; cerebrospinal fluids
13.  Mass spectrometry-based N-glycoproteomics for cancer biomarker discovery 
Clinical proteomics  2014;11(1):18.
Glycosylation is estimated to be found in over 50% of human proteins. Aberrant protein glycosylation and alteration of glycans are closely related to many diseases. More than half of the cancer biomarkers are glycosylated-proteins, and specific glycoforms of glycosylated-proteins may serve as biomarkers for either the early detection of disease or the evaluation of therapeutic efficacy for treatment of diseases. Glycoproteomics, therefore, becomes an emerging field that can make unique contributions to the discovery of biomarkers of cancers. The recent advances in mass spectrometry (MS)-based glycoproteomics, which can analyze thousands of glycosylated-proteins in a single experiment, have shown great promise for this purpose. Herein, we described the MS-based strategies that are available for glycoproteomics, and discussed the sensitivity and high throughput in both qualitative and quantitative manners. The discovery of glycosylated-proteins as biomarkers in some representative diseases by employing glycoproteomics was also summarized.
doi:10.1186/1559-0275-11-18
PMCID: PMC4017703  PMID: 24872809
Glycosylation; Disease proteomics; Mass spectrometry
14.  Glycoproteomics enabled by tagging sialic acid- or galactose-terminated glycans 
Glycobiology  2012;23(2):211-221.
In this paper, we present two complementary strategies for enrichment of glycoproteins on living cells that combine the desirable attributes of “robust enrichment” afforded by covalent-labeling techniques and “specificity for glycoproteins” typically provided by lectin or antibody affinity reagents. Our strategy involves the selective introduction of aldehydes either into sialic acids by periodate oxidation (periodate oxidation and aniline-catalyzed oxime ligation (PAL)) or into terminal galactose and N-acetylgalactosamine residues by galactose oxidase (galactose oxidase and aniline-catalyzed oxime ligation (GAL)), followed by aniline-catalyzed oxime ligation with aminooxy-biotin to biotinylate the glycans of glycoprotein subpopulations with high efficiency and cell viability. As expected, the two methods exhibit reciprocal tagging efficiencies when applied to fully sialylated cells compared with sialic acid-deficient cells. To assess the utility of these labeling methods for glycoproteomics, we enriched the PAL- and GAL-labeled (biotinylated) glycoproteome by adsorption onto immobilized streptavidin. Glycoprotein identities (IDs) and N-glycosylation site information were then obtained by liquid chromatography-tandem mass spectrometry on total tryptic peptides and on peptides subsequently released from N-glycans still bound to the beads using peptide N-glycosidase F. A total of 175 unique N-glycosylation sites were identified, belonging to 108 nonredundant glycoproteins. Of the 108 glycoproteins, 48 were identified by both methods of labeling and the remainder was identified using PAL on sialylated cells (40) or GAL on sialic acid-deficient cells (20). Our results demonstrate that PAL and GAL can be employed as complementary methods of chemical tagging for targeted proteomics of glycoprotein subpopulations and identification of glycosylation sites of proteins on cells with an altered sialylation status.
doi:10.1093/glycob/cws144
PMCID: PMC3531297  PMID: 23070960
aniline catalysis; galactose oxidase; glycoproteomics; mass spectrometry; oxime ligation; periodate oxidation
15.  Large-scale quantitative glycoproteomics analysis of site-specific glycosylation occupancy† 
Molecular bioSystems  2012;8(11):2850-2856.
Disease-associated aberrant glycosylation may be protein specific and glycosylation site specific. Quantitative assessment of glycosylation changes at a site-specific molecular level may represent one of the initial steps for systematically revealing the glycosylation abnormalities associated with a disease or biological state. Comparative quantitative profiling of glycoproteome to provide accurate quantification of site-specific glycosylation occupancy has been a challenging task, requiring a concerted approach drawing from a variety of techniques. In this report, we present a quantitative glycoproteomics method that allows global scale identification and comparative quantification of glycosylation site occupancy using mass spectrometry. We further demonstrated this approach by quantitatively characterizing the N-glycoproteome of human pancreas.
doi:10.1039/c2mb25268f
PMCID: PMC3463725  PMID: 22892896
16.  The quantitative proteomes of human-induced pluripotent stem cells and embryonic stem cells 
An in-depth proteomic comparison of human-induced pluripotent stem cells, and their parent fibroblast cells, with embryonic stem cells shows that the reprogramming process comprehensively remodels protein expression levels, creating cells that closely resemble natural stem cells.
We present here a large proteomic characterization of human embryonic stem cells, human-induced pluripotent stem cells and their parental fibroblasts cell lines.Overall, 97.8% of the 2683 quantified proteins in four experiments showed no significant differences in abundance between hESC and hiPSC highlighting the high similarity of these pluripotent cell lines.In total, 58 proteins were found significantly differentially expressed between hiPSCs and hESCs. The observed low overlap of these proteins with previous transcriptomic studies suggests that those differences do no reflect a recurrent molecular signature.
Human embryonic stem cells (hESCs) are capable of self-renewal and multi-lineage differentiation. However, the use of hESCs for clinical treatment entails ethical issues as they are derived from human embryos. Recently, reprogramming of somatic cells to an embryonic stem cell-like state, named induced pluripotent stem cells (iPSCs), was achieved through ectopic expression of defined factors. In addition to their clinical potential, hiPSCs represent a unique tool to develop cellular models for human diseases as well. Although current functional assays (e.g., tetraploid complementation) have confirmed the pluripotency of hiPSCs, there might still be significant differences (e.g., differentiation potential) when compared with their natural hESCs counterparts. Consequently, an extensive molecular characterization to address differences and similarities between these two pluripotent cell lines seems to be a prerequisite before any clinical application is conducted. Despite that great efforts, mainly at the genomic levels, have been made to address how similar hESCs and hiPSCs are, the definite answer to this fundamental question is currently still debated. Direct assessment of protein levels has yet to be incorporated into these integrative systems-level analyses. Protein levels are tuned by intricate mechanisms of gene expression regulation and it has recently been documented that mRNA and protein levels poorly correlate in mouse ESCs. Here, we use in-depth quantitative proteomics to gain insights into the differences and similarities in the protein content of two hiPS cell lines, their precursor IMR90 and 4Skin fibroblast cell lines and one hES cell line, providing novel molecular signatures that may assist in filling a gap in the understanding of pluripotency.
To study the degree of similarity, at the protein level, between hiPSCs and hESCs, four MS-based proteomic experiments were designed that use our in-house developed triplex dimethyl labeling chemistry followed by extensive fractionation by strong cation exchange (SCX) chromatography to reduce the sample complexity. High-resolution LC-MS/MS with dedicated fragmentation schemes (i.e., electron transfer dissociation, collision-induced dissociation and higher-energy collision dissociation) was subsequently used to maximize peptide identification rates. A total of 348 LC-MS/MS analyses (including technical and biological replicates) were performed. We confidently identified 1 593 446 peptide spectrum matches (peptide FDR<1%) corresponding to 10 628 unique protein groups (protein FDR∼4%). Using the extracted ion chromatograms, we also estimated the absolute abundance of the proteins within the samples spanning six orders of magnitude. To the best of our knowledge, the coverage obtained in this study represents the largest achieved by any proteomics screen on pluripotent cells.
Most importantly, our results indicate that the reprogramming process remodeled the proteome of both fibroblast cell lines to a profile that closely resembles the pluripotent hESCs proteome: 97.8% of the quantified proteins (2638 proteins in all four experiments) showed nonsignificant changes. Nevertheless, a small fraction of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly regulated between hiPSCs and hESCs. A comparison of the regulated proteins to previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature. On the other side, the inclusion of the two parental fibroblast cell lines in our analysis allowed us to study changes in the proteome at both the starting and end points of the reprogramming process. As expected, the vast majority of the proteins (73.4%) showed differential expression between the parental fibroblasts and the reprogrammed pluripotent cells.
To find out if the differences observed in our study were a consequence of transcriptional or translational regulation, we performed paired genome-wide gene expression analyses on the same six samples that were used for the proteomic profiling. Overall, we observed a good correlation between mRNA and protein levels (r∼0.7). These results further authenticated the proteomic measurements and implied a high degree of control at the transcriptional level. Nevertheless, numerous genes were found uncorrelated highlighting the necessity of complementing transcriptomic-based approaches with proteomics.
Assessing relevant molecular differences between human-induced pluripotent stem cells (hiPSCs) and human embryonic stem cells (hESCs) is important, given that such differences may impact their potential therapeutic use. Controversy surrounds recent gene expression studies comparing hiPSCs and hESCs. Here, we present an in-depth quantitative mass spectrometry-based analysis of hESCs, two different hiPSCs and their precursor fibroblast cell lines. Our comparisons confirmed the high similarity of hESCs and hiPSCS at the proteome level as 97.8% of the proteins were found unchanged. Nevertheless, a small group of 58 proteins, mainly related to metabolism, antigen processing and cell adhesion, was found significantly differentially expressed between hiPSCs and hESCs. A comparison of the regulated proteins with previously published transcriptomic studies showed a low overlap, highlighting the emerging notion that differences between both pluripotent cell lines rather reflect experimental conditions than a recurrent molecular signature.
doi:10.1038/msb.2011.84
PMCID: PMC3261715  PMID: 22108792
human embryonic stem cells; human-induced pluripotent stem cells; proteomics; quantitation
17.  N-Glycoproteome of E14.Tg2a Mouse Embryonic Stem Cells 
PLoS ONE  2013;8(2):e55722.
E14.Tg2a mouse embryonic stem (mES) cells are a widely used host in gene trap and gene targeting techniques. Molecular characterization of host cells will provide background information for a better understanding of functions of the knockout genes. Using a highly selective glycopeptide-capture approach but ordinary liquid chromatography coupled mass spectrometry (LC-MS), we characterized the N-glycoproteins of E14.Tg2a cells and analyzed the close relationship between the obtained N-glycoproteome and cell-surface proteomes. Our results provide a global view of cell surface protein molecular properties, in which receptors seem to be much more diverse but lower in abundance than transporters on average. In addition, our results provide a systematic view of the E14.Tg2a N-glycosylation, from which we discovered some striking patterns, including an evolutionarily preserved and maybe functionally selected complementarity between N-glycosylation and the transmembrane structure in protein sequences. We also observed an environmentally influenced N-glycosylation pattern among glycoenzymes and extracellular matrix proteins. We hope that the acquired information enhances our molecular understanding of mES E14.Tg2a as well as the biological roles played by N-glycosylation in cell biology in general.
doi:10.1371/journal.pone.0055722
PMCID: PMC3565968  PMID: 23405203
18.  Analysis of Protein Glycosylation and Phosphorylation Using Liquid Phase Separation, Protein Microarray Technology, and Mass Spectrometry 
Summary
Protein glycosylation and phosphorylation are very common posttranslational modifications. The alteration of these modifications in cancer cells is closely related to the onset and progression of cancer and other disease states. In this protocol, strategies for monitoring the changes in protein glycosylation and phosphorylation in serum or tissue cells on a global scale and specifically characterizing these alterations are included. The technique is based on lectin affinity enrichment for glycoproteins, all liquid-phase two-dimensional fractionation, protein microarray, and mass spectrometry technology. Proteins are separated based on pI in the first dimension using chromatofocusing (CF) or liquid isoelectric focusing (IEF) followed by the second-dimension separation using nonporous silica RP-HPLC. Five lectins with different binding specificities to glycan structures are used for screening glycosylation patterns in human serum through a biotin–streptavidin system. Fluorescent phosphodyes and phosphospecific antibodies are employed to detect specific phosphorylated proteins in cell lines or human tissues. The purified proteins of interest are identified by peptide sequencing. Their modifications including glycosylation and phosphorylation could be further characterized by mass-spectrometry-based approaches. These strategies can be used in biological samples for large-scale glycoproteome/phosphoproteome screening as well as for individual protein modification analysis.
doi:10.1007/978-1-59745-493-3_20
PMCID: PMC2921194  PMID: 19241043
Glycosylation; Phosphorylation; Posttranslational modification; Protein microarrays; Liquid phase separation; Lectin; Mass spectrometry
19.  N-Glycomic and -Glycoproteomic Studies in the Social Amoebae 
Summary
N-glycans modify the great majority of all secreted and plasma membrane proteins, which themselves constitute one-third to one-half of the proteome. The ultimate definition of the glycoproteome would be the identification of all the N-glycans attached to all the modified asparaginyl sites of all the proteins, but glycosylation heterogeneity makes this an unachievable goal. However, mass spectrometry in combination with other methods does have the power to deeply mine the N-glycome of Dictyostelium, and characterize glycan profiles at individual sites of glycoproteins. Recent studies from our laboratories using mass spectrometry-based methods have confirmed basic precepts of the N-glycome based on prior classical methods using radiotracer methods, and have extended the scope of glycan diversity and the distribution of glycan types across specific glycoprotein attachment sites. The protocols described here simplify studies of the N-glycome and -glycoproteome, which should prove useful for interpreting mutant phenotypes, conducting interstrain and interspecies comparisons, and investigating glycan functions in glycoproteins of interest.
doi:10.1007/978-1-62703-302-2_11
PMCID: PMC4060149  PMID: 23494309
Dictyostelium; glycosylation; glycome; glycoproteome; mass spectrometry; phosphoglycans; sulfated glycans
20.  A Novel Workflow for Glycopeptide Analysis Using Cellulose-Based Separation Cartridges, TMT-Labeling and LTQ Orbitrap ETD 
RP-16
Currently, the field of glycoproteomics is focused on development of methods aimed at sensitive detection of glycans and various glycoconjugates, with particular emphasis on accurate quantitative analysis and detailed structural characterization. In this work, we describe a novel workflow, which combines Tandem mass tags (TMT) labeling and a new method for efficient extraction of glycopeptides from a complex digest mixture with nearly negligible loss of glycopeptide material or carryover and subsequent ETD analysis. Based on previously published work on the use of cellulose for isolation of glycopeptides from complex proteolytic digests in solution, we examined the use of this inexpensive material in a column format. An in-house column was designed that incorporated cellulose, and C-18 packing material. Initial method was developed using alpha 1 acid glycoprotein, due to their high glycan content and they carry sialic acid, a biologically significant monosaccharide unit. The method was extended to bovine fetuin to examine the efficiency of our extraction method for O-linked glycopeptides. Bovine fetuin is a well-characterized glycoprotein containing both N- and O-linked sites of glycosylation. Additional validation were done with more complex standard protein digest mixtures. The addition of the basic TMT labels increased the average charge state of the precursors and as a result improves ETD fragmentation of acidic glycopeptides. Furthermore, these tags provide a means for relative quantification of labile PTMs. A simple and efficient method was developed for on-column isolation of glycopeptides from complex proteolytic digests, using cellulose as solid-phase extraction material. TMT labeled N- and O-linked glycopeptides (of varying length) were enriched in good yield for further glycoproteomics studies. Combination of HCD/ETD MS/MS analysis provides optimal information for both sequencing and localization of modifcation site on the glycopeptides. Aiming at replacing or complementing lectin-based purification methods, this technique is extremely simple, cost-effective, and efficient.
PMCID: PMC2918030
21.  Automated glycopeptide analysis—review of current state and future directions 
Briefings in Bioinformatics  2012;14(3):361-374.
Glycosylation of proteins is involved in immune defense, cell–cell adhesion, cellular recognition and pathogen binding and is one of the most common and complex post-translational modifications. Science is still struggling to assign detailed mechanisms and functions to this form of conjugation. Even the structural analysis of glycoproteins—glycoproteomics—remains in its infancy due to the scarcity of high-throughput analytical platforms capable of determining glycopeptide composition and structure, especially platforms for complex biological mixtures. Glycopeptide composition and structure can be determined with high mass-accuracy mass spectrometry, particularly when combined with chromatographic separation, but the sheer volume of generated data necessitates computational software for interpretation. This review discusses the current state of glycopeptide assignment software—advances made to date and issues that remain to be addressed. The various software and algorithms developed so far provide important insights into glycoproteomics. However, there is currently no freely available software that can analyze spectral data in batch and unambiguously determine glycopeptide compositions for N- and O-linked glycopeptides from relevant biological sources such as human milk and serum. Few programs are capable of aiding in structural determination of the glycan component. To significantly advance the field of glycoproteomics, analytical software and algorithms are required that: (i) solve for both N- and O-linked glycopeptide compositions, structures and glycosites in biological mixtures; (ii) are high-throughput and process data in batches; (iii) can interpret mass spectral data from a variety of sources and (iv) are open source and freely available.
doi:10.1093/bib/bbs045
PMCID: PMC3659302  PMID: 22843980
glycopeptide; glycoproteomics; glycopeptidomics; bioinformatics; N-linked; O-linked
22.  Analysis of age and gender associated N-glycoproteome in human whole saliva 
Clinical proteomics  2014;11(1):25.
Background
Glycoproteins comprise a large portion of the salivary proteome and have great potential for biomarker discovery and disease diagnosis. However, the rate of production and the concentration of whole saliva change with age, gender and physiological states of the human body. Therefore, a thorough understanding of the salivary glycoproteome of healthy individuals of different ages and genders is a prerequisite for saliva to have clinical utility.
Methods
Formerly N-linked glycopeptides were isolated from the pooled whole saliva of six age and gender groups by hydrazide chemistry and hydrophilic affinity methods followed by mass spectrometry identification. Selected physiochemical characteristics of salivary glycoproteins were analyzed, and the salivary glycoproteomes of different age and gender groups were compared based on their glycoprotein components and gene ontology.
Results and discussion
Among 85 N-glycoproteins identified in healthy human saliva, the majority were acidic proteins with low molecular weight. The numbers of salivary N-glycoproteins increased with age. Fifteen salivary glycoproteins were identified as potential age- or gender-associated glycoproteins, and many of them have functions related to innate immunity against microorganisms and oral cavity protection. Moreover, many salivary glycoproteins have been previously reported as disease related glycoproteins. This study reveals the important role of salivary glycoproteins in the maintenance of oral health and homeostasis and the great potential of saliva for biomarker discovery and disease diagnosis.
doi:10.1186/1559-0275-11-25
PMCID: PMC4070402  PMID: 24994967
Saliva; Glycoproteome; Glycoproteins; Age; Gender; Hydrazide chemistry; Hydrophilic affinity; Mass spectrometry
23.  Chemical Approaches to Perturb, Profile, and Perceive Glycans 
Accounts of chemical research  2009;42(6):788-797.
Conspectus
Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, as their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenging to routine practice.
In studies of proteins and nucleic acids, functional studies have often relied on genetic manipulations to perturb structure. Though not directly subject to mutation, we can determine glycan structure-function relationships by synthesizing defined glycoconjugates or by altering natural glycosylation pathways. Chemical syntheses of uniform glycoproteins and polymeric glycoprotein mimics have facilitated the study of individual glycoconjugates in the absence of glycan microheterogeneity. Alternatively, selective inhibition or activation of glycosyltransferases or glycosidases can define the biological roles of the corresponding glycans. Investigators have developed tools including small molecule inhibitors, decoy substrates, and engineered-proteins to modify cellular glycans. Current approaches offer a precision approaching that of genetic control.
Genomic and proteomic profiling form a basis for biological discovery. Glycans also present a rich matrix of information that adapts rapidly to changing environs. Glycomic and glycoproteomic analyses via microarrays and mass spectrometry are beginning to characterize alterations in glycans that correlate with disease. These approaches have already identified several cancer biomarkers. Metabolic labeling can identify recently synthesized glycans and thus directly track glycan dynamics. This approach can highlight changes in physiology or environment and may be more informative than steady-state analyses. Together, glycomic and metabolic labeling techniques provide a comprehensive description of glycosylation as a foundation for hypothesis generation.
Direct visualization of proteins via the green fluorescent protein (GFP) and its congeners has revolutionized the field of protein dynamics. Similarly, the ability to perceive the spatial organization of glycans could transform our understanding of their role in development, infection, and disease progression. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. These results have highlighted the need for additional imaging probes.
doi:10.1021/ar800267j
PMCID: PMC2697281  PMID: 19361192
24.  Chemical Approaches To Perturb, Profile, and Perceive Glycans 
Accounts of Chemical Research  2009;42(6):788-797.
Glycosylation is an essential form of post-translational modification that regulates intracellular and extracellular processes. Regrettably, conventional biochemical and genetic methods often fall short for the study of glycans, because their structures are often not precisely defined at the genetic level. To address this deficiency, chemists have developed technologies to perturb glycan biosynthesis, profile their presentation at the systems level, and perceive their spatial distribution. These tools have identified potential disease biomarkers and ways to monitor dynamic changes to the glycome in living organisms. Still, glycosylation remains the underexplored frontier of many biological systems. In this Account, we focus on research in our laboratory that seeks to transform the study of glycan function from a challenge to routine practice.
In studies of proteins and nucleic acids, functional studies have often relied on genetic manipulations to perturb structure. Though not directly subject to mutation, we can determine glycan structure−function relationships by synthesizing defined glycoconjugates or by altering natural glycosylation pathways. Chemical syntheses of uniform glycoproteins and polymeric glycoprotein mimics have facilitated the study of individual glycoconjugates in the absence of glycan microheterogeneity. Alternatively, selective inhibition or activation of glycosyltransferases or glycosidases can define the biological roles of the corresponding glycans. Investigators have developed tools including small molecule inhibitors, decoy substrates, and engineered proteins to modify cellular glycans. Current approaches offer a precision approaching that of genetic control.
Genomic and proteomic profiling form a basis for biological discovery. Glycans also present a rich matrix of information that adapts rapidly to changing environs. Glycomic and glycoproteomic analyses via microarrays and mass spectrometry are beginning to characterize alterations in glycans that correlate with disease. These approaches have already identified several cancer biomarkers. Metabolic labeling can identify recently synthesized glycans and thus directly track glycan dynamics. This approach can highlight changes in physiology or environment and may be more informative than steady-state analyses. Together, glycomic and metabolic labeling techniques provide a comprehensive description of glycosylation as a foundation for hypothesis generation.
Direct visualization of proteins via the green fluorescent protein (GFP) and its congeners has revolutionized the field of protein dynamics. Similarly, the ability to perceive the spatial organization of glycans could transform our understanding of their role in development, infection, and disease progression. Fluorescent tagging in cultured cells and developing organisms has revealed important insights into the dynamics of these structures during growth and development. These results have highlighted the need for additional imaging probes.
doi:10.1021/ar800267j
PMCID: PMC2697281  PMID: 19361192
25.  Towards an integrated proteomic and glycomic approach to finding cancer biomarkers 
Genome Medicine  2009;1(6):57.
Advances in mass spectrometry have had a great impact on the field of proteomics. A major challenge of proteomic analysis has been the elucidation of glycan modifications of proteins in complex proteomes. Glycosylation is the most structurally elaborate and diverse type of protein post-translational modification and, because of this, proteomics and glycomics have largely developed independently. However, given that such a large proportion of proteins contain glycan modifications, and that these may be important for their function or may produce biologically relevant protein variation, a convergence of the fields of glycomics and proteomics would be highly desirable. Here we review the current status of glycoproteomic efforts, focusing on the identification of glycoproteins as cancer biomarkers.
doi:10.1186/gm57
PMCID: PMC2703866  PMID: 19519948

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