Recent advances in methods for the analysis of protein o-glycosylation at proteome level.

O-Glycosylation, which refers to the glycosylation of the hydroxyl group of side chains of Serine/Threonine/Tyrosine residues, is one of the most common post-translational modifications. Compared with N-linked glycosylation, O-glycosylation is less explored because of its complex structure and relatively low abundance. Recently, O-glycosylation has drawn more and more attention for its various functions in many sophisticated biological processes. To obtain a deep understanding of O-glycosylation, many efforts have been devoted to develop effective strategies to analyze the two most abundant types of O-glycosylation, i.e. O-N-acetylgalactosamine and O-N-acetylglucosamine glycosylation. In this review, we summarize the proteomics workflows to analyze these two types of O-glycosylation. For the large-scale analysis of mucin-type glycosylation, the glycan simplification strategies including the ''SimpleCell'' technology were introduced. A variety of enrichment methods including lectin affinity chromatography, hydrophilic interaction chromatography, hydrazide chemistry, and chemoenzymatic method were introduced for the proteomics analysis of O-N-acetylgalactosamine and O-N-acetylglucosamine glycosylation.

[1]  H. Zou,et al.  Proteomics Analysis of O-GalNAc Glycosylation in Human Serum by an Integrated Strategy. , 2017, Analytical chemistry.

[2]  J. L. Asensio,et al.  Serine versus threonine glycosylation with α-O-GalNAc: unexpected selectivity in their molecular recognition with lectins. , 2014, Chemistry.

[3]  Yoshiki Narimatsu,et al.  The GalNAc-type O-Glycoproteome of CHO Cells Characterized by the SimpleCell Strategy* , 2014, Molecular & Cellular Proteomics.

[4]  Jennifer A. Prescher,et al.  Chemistry in living systems , 2005, Nature chemical biology.

[5]  V. Piller,et al.  Changes in Metabolic Chemical Reporter Structure Yield a Selective Probe of O-GlcNAc Modification , 2014, Journal of the American Chemical Society.

[6]  G. Hart,et al.  Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins. , 1997, Annual review of biochemistry.

[7]  Pamela Greenwell,et al.  The lectin Helix pomatia agglutinin recognizes O-GlcNAc containing glycoproteins in human breast cancer. , 2012, Glycobiology.

[8]  C. Srisomsap,et al.  Proteomic analysis and abrogated expression of O‐GlcNAcylated proteins associated with primary breast cancer , 2013, Proteomics.

[9]  Carolyn R Bertozzi,et al.  A chemical approach for identifying O-GlcNAc-modified proteins in cells , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[10]  G. Hart,et al.  O-Glycosylation of Nuclear and Cytosolic Proteins , 2000, The Journal of Biological Chemistry.

[11]  Lance Wells,et al.  Mapping Sites of O-GlcNAc Modification Using Affinity Tags for Serine and Threonine Post-translational Modifications* , 2002, Molecular & Cellular Proteomics.

[12]  G. Larson,et al.  LC-MS/MS characterization of O-glycosylation sites and glycan structures of human cerebrospinal fluid glycoproteins. , 2013, Journal of proteome research.

[13]  G. Hart,et al.  Site-Specific GlcNAcylation of Human Erythrocyte Proteins , 2009, Diabetes.

[14]  Jonathan C Trinidad,et al.  O-Linked N-Acetylglucosamine Proteomics of Postsynaptic Density Preparations Using Lectin Weak Affinity Chromatography and Mass Spectrometry*S , 2006, Molecular & Cellular Proteomics.

[15]  M. Wolfert,et al.  Glycopeptide-specific monoclonal antibodies suggest new roles for O-GlcNAc. , 2010, Nature chemical biology.

[16]  Russell A. Reeves,et al.  Characterization of the specificity of O-GlcNAc reactive antibodies under conditions of starvation and stress. , 2014, Analytical biochemistry.

[17]  R. Aebersold,et al.  Selected reaction monitoring–based proteomics: workflows, potential, pitfalls and future directions , 2012, Nature Methods.

[18]  K. Medzihradszky,et al.  How to Dig Deeper? Improved Enrichment Methods for Mucin Core-1 Type Glycopeptides* , 2012, Molecular & Cellular Proteomics.

[19]  G. J. Rademaker,et al.  Determination of glycosylation sites in O-linked glycopeptides: a sensitive mass spectrometric protocol , 1993 .

[20]  T. Lefebvre,et al.  Characterization of O-GlcNAc cycling and proteomic identification of differentially O-GlcNAcylated proteins during G1/S transition. , 2012, Biochimica et biophysica acta.

[21]  B. Sarg,et al.  Evaluation of non‐reductive β‐elimination/Michael addition for glycosylation site determination in mucin‐like O‐glycopeptides , 2011, Electrophoresis.

[22]  Matthew E Monroe,et al.  Tandem mass spectrometry identifies many mouse brain O-GlcNAcylated proteins including EGF domain-specific O-GlcNAc transferase targets , 2012, Proceedings of the National Academy of Sciences.

[23]  B. Garcia,et al.  A Novel Quantitative Mass Spectrometry Platform for Determining Protein O-GlcNAcylation Dynamics* , 2016, Molecular & Cellular Proteomics.

[24]  K. Medzihradszky,et al.  O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum , 2016, Glycoconjugate Journal.

[25]  Robert J Chalkley,et al.  Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides , 2009, Proceedings of the National Academy of Sciences.

[26]  Shinichi Iwamoto,et al.  Reversible hydrazide chemistry-based enrichment for O-GlcNAc-modified peptides and glycopeptides having non-reducing GlcNAc residues. , 2013, The Analyst.

[27]  H. Wandall,et al.  Mining the O-glycoproteome using zinc-finger nuclease–glycoengineered SimpleCell lines , 2011, Nature Methods.

[28]  C. Bertozzi,et al.  Isotope-targeted glycoproteomics (IsoTaG): a mass-independent platform for intact N- and O-glycopeptide discovery and analysis , 2015, Nature Methods.

[29]  C. Hesse,et al.  Enrichment of glycopeptides for glycan structure and attachment site identification , 2009, Nature Methods.

[30]  Chong Yu,et al.  A metabolic labeling approach toward proteomic analysis of mucin-type O-linked glycosylation , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[31]  E. Bennett,et al.  Enhanced Mass Spectrometric Mapping of the Human GalNAc-type O-Glycoproteome with SimpleCells* , 2013, Molecular & Cellular Proteomics.

[32]  Zhihong Guo,et al.  Combination of beta-elimination and liquid chromatography/quadrupole time-of-flight mass spectrometry for the determination of O-glycosylation sites. , 2009, Talanta.

[33]  G. Hart,et al.  O-GlcNAc Cycling Enzymes Associate with the Translational Machinery and Modify Core Ribosomal Proteins , 2010, Molecular biology of the cell.

[34]  Jonas Nilsson,et al.  Human Urinary Glycoproteomics; Attachment Site Specific Analysis of N- and O-Linked Glycosylations by CID and ECD* , 2011, Molecular & Cellular Proteomics.

[35]  L. McMahon,et al.  O-GlcNAcylation and neurodegeneration , 2017, Brain Research Bulletin.

[36]  K. Medzihradszky,et al.  Affinity Enrichment and Characterization of Mucin Core-1 Type Glycopeptides from Bovine Serum* , 2009, Molecular & Cellular Proteomics.

[37]  H. Hang,et al.  Chemical reporters for fluorescent detection and identification of O-GlcNAc-modified proteins reveal glycosylation of the ubiquitin ligase NEDD4-1 , 2011, Proceedings of the National Academy of Sciences.

[38]  L. Gerace,et al.  Monoclonal antibodies identify a group of nuclear pore complex glycoproteins , 1987, The Journal of cell biology.

[39]  Ronghu Wu,et al.  Specific Identification of Glycoproteins Bearing the Tn Antigen in Human Cells. , 2017, Angewandte Chemie.

[40]  G. Hart,et al.  Nuclear pore complex glycoproteins contain cytoplasmically disposed O- linked N-acetylglucosamine , 1987, The Journal of cell biology.

[41]  G. Boons,et al.  Synthetic Receptors for the High‐Affinity Recognition of O‐GlcNAc Derivatives , 2016, Angewandte Chemie.

[42]  M. Larsen,et al.  Purification and identification of O-GlcNAc-modified peptides using phosphate-based alkyne CLICK chemistry in combination with titanium dioxide chromatography and mass spectrometry. , 2011, Journal of proteome research.

[43]  Gerald W. Hart,et al.  Analysis of Protein O‐GlcNAcylation by Mass Spectrometry , 2017, Current protocols in protein science.

[44]  Jennifer A. Prescher,et al.  Copper-free click chemistry in living animals , 2010, Proceedings of the National Academy of Sciences.

[45]  D. V. van Aalten,et al.  A mutant O-GlcNAcase enriches Drosophila developmental regulators. , 2017, Nature chemical biology.

[46]  A. Burlingame,et al.  Polycomb repressive complex 2 is necessary for the normal site-specific O-GlcNAc distribution in mouse embryonic stem cells , 2011, Proceedings of the National Academy of Sciences.

[47]  J. Shabanowitz,et al.  Enrichment and Site Mapping of O-Linked N-Acetylglucosamine by a Combination of Chemical/Enzymatic Tagging, Photochemical Cleavage, and Electron Transfer Dissociation Mass Spectrometry* , 2009, Molecular & Cellular Proteomics.

[48]  C. Vicent,et al.  A synthetic lectin for O-linked beta-N-acetylglucosamine. , 2009, Angewandte Chemie.

[49]  G. Hart,et al.  Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. , 2001, Analytical biochemistry.

[50]  I. Landrieu,et al.  Identification of O-GlcNAc sites within peptides of the Tau protein and their impact on phosphorylation. , 2011, Molecular bioSystems.

[51]  Lance Wells,et al.  Combining high-energy C-trap dissociation and electron transfer dissociation for protein O-GlcNAc modification site assignment. , 2011, Journal of proteome research.

[52]  A. Edge.  Deglycosylation of glycoproteins with trifluoromethanesulphonic acid: elucidation of molecular structure and function. , 2003, The Biochemical journal.

[53]  Susana Comte-Walters,et al.  Identification of O-Linked N-Acetylglucosamine (O-GlcNAc)-modified Osteoblast Proteins by Electron Transfer Dissociation Tandem Mass Spectrometry Reveals Proteins Critical for Bone Formation* , 2013, Molecular & Cellular Proteomics.

[54]  N. Greenspan,et al.  Cytologic assessment of nuclear and cytoplasmic O-linked N-acetylglucosamine distribution by using anti-streptococcal monoclonal antibodies. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[55]  A. Pandey,et al.  The dynamic stress-induced “O-GlcNAc-ome” highlights functions for O-GlcNAc in regulating DNA damage/repair and other cellular pathways , 2011, Amino Acids.

[56]  A. Pandey,et al.  Dynamic Interplay between O-Linked N-Acetylglucosaminylation and Glycogen Synthase Kinase-3-dependent Phosphorylation* , 2007, Molecular & Cellular Proteomics.

[57]  K. Medzihradszky,et al.  Enrichment of O-GlcNAc modified proteins by the periodate oxidation-hydrazide resin capture approach. , 2010, Journal of proteome research.

[58]  Bernhard Kuster,et al.  Discovery of O-GlcNAc-modified Proteins in Published Large-scale Proteome Data* , 2012, Molecular & Cellular Proteomics.

[59]  S. Vakhrushev,et al.  Probing the O-Glycoproteome of Gastric Cancer Cell Lines for Biomarker Discovery* , 2015, Molecular & Cellular Proteomics.

[60]  M. Fukuda.  Roles of mucin-type O-glycans in cell adhesion. , 2002, Biochimica et biophysica acta.

[61]  Adnan Halim,et al.  Advances in mass spectrometry driven O-glycoproteomics. , 2015, Biochimica et biophysica acta.

[62]  R. Roeder,et al.  GlcNAcylation of histone H2B facilitates its monoubiquitination , 2011, Nature.

[63]  Animesh Nandi,et al.  Global identification of O-GlcNAc-modified proteins. , 2006, Analytical chemistry.

[64]  Christoph Wagener,et al.  COSMC knockdown mediated aberrant O-glycosylation promotes oncogenic properties in pancreatic cancer , 2015, Molecular Cancer.

[65]  S. Brunak,et al.  A Systematic Study of Site-specific GalNAc-type O-Glycosylation Modulating Proprotein Convertase Processing* , 2011, The Journal of Biological Chemistry.

[66]  P. Kloetzel,et al.  novel Biotin-Cystamine Tag , 2012 .

[67]  S. Brunak,et al.  Precision mapping of the human O‐GalNAc glycoproteome through SimpleCell technology , 2013, The EMBO journal.

[68]  A. Burlingame,et al.  Global Identification and Characterization of Both O-GlcNAcylation and Phosphorylation at the Murine Synapse* , 2012, Molecular & Cellular Proteomics.

[69]  R. Angeletti,et al.  Optimization of the β-elimination/michael addition chemistry on reversed-phase supports for mass spectrometry analysis of O-linked protein modifications. , 2013, Journal of Biomolecular Techniques.

[70]  Jennifer J. Kohler,et al.  Metabolic cross-talk allows labeling of O-linked β-N-acetylglucosamine-modified proteins via the N-acetylgalactosamine salvage pathway , 2011, Proceedings of the National Academy of Sciences.

[71]  Linda C Hsieh-Wilson,et al.  A chemoenzymatic approach toward the rapid and sensitive detection of O-GlcNAc posttranslational modifications. , 2003, Journal of the American Chemical Society.

[72]  K. G. Hagen,et al.  O-Glycosylation and Development , 2014 .

[73]  Yingming Zhao,et al.  Tagging-via-substrate strategy for probing O-GlcNAc modified proteins. , 2005, Journal of proteome research.

[74]  Adnan Halim,et al.  Characterizing the O-glycosylation landscape of human plasma, platelets, and endothelial cells. , 2017, Blood advances.

[75]  Sarah Tisdale,et al.  A functional RNAi screen links O-GlcNAc modification of ribosomal proteins to stress granule and processing body assembly , 2008, Nature Cell Biology.

[76]  B. Kuster,et al.  Proteome Wide Purification and Identification of O‑glcnac-modified Proteins Using Click Chemistry and Mass Spectrometry , 2022 .

[77]  Guangshun Wang,et al.  A novel strategy for global mapping of O-GlcNAc proteins and peptides using selective enzymatic deglycosylation, HILIC enrichment and mass spectrometry identification. , 2017, Talanta.