Glycomic Approaches to Study GlcNAcylation: Protein Identification, Site-mapping, and Site-specific O-GlcNAc Quantitation
O-Linked β-N-acetylglucosamine (O-GlcNAc) is an enzyme-catalyzed posttranslational modification of serine or threonine side chains of nuclear and cytoplasmic proteins. O-GlcNAc is present in all metazoans and in viruses that infect eukaryotic cells. GlcNAcylation is dynamic and has a high cycling rate on many proteins in response to cellular metabolism and various environmental stimuli. The rapid cycling of O-GlcNAc modulates many biological processes, including transcriptional regulation, stress responses, cell cycle regulation, and protein synthesis and turnover.
Despite the importance of O-GlcNAc, progress during the past two decades in this field has been slow. One of the major obstacles is the lack of simple and sensitive tools for efficient O-GlcNAc detection and localization. Recently developed O-GlcNAc derivatization and enrichment approaches, together with new techniques in mass spectrometric instrumentation and methods, have provided breakthroughs in O-GlcNAc site localization and site-specific quantitation. In this review, we will discuss how the current techniques are expanding our knowledge about O-GlcNAc proteomics/glycomics and functions.
- Torres C-R, Hart GW. Topography and polypeptide distribution of terminal N-acetylglucosamine residues on the surfaces of intact lymphocytes. Evidence for O-linked GlcNAc. J Biol Chem 1984;259:3308–17.
- Holt G, Hart GW. Subcellular distribution of terminal N-acetylglucosamine residues on oligosaccharides. Distribution of the novel protein-saccharide structure—O-linked GlcNAc. J Biol Chem 1986;261:8049–57.
- Hart G, Housley M, Slawson C. Cycling of O-linked b-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 2007;446:1017–22. CrossRef
- McClain D, Crook E. Hexosamines and insulin resistance. Diabetes 1996;45:1003–9. CrossRef
- Manning G, Whyte DB, Martinez R, Hunter T, Sudarsanam S. The protein kinase complement of the human genome. Science 2002;298:1912–34. CrossRef
- Kreppel L, Hart G. Regulation of a cytosolic and nuclear O-GlcNAc transferase. Role of the tetratricopeptide repeats. J Biol Chem 1999;274:32015–22. CrossRef
- Lubas W, Hanover J. Functional expression of O-linked GlcNAc transferase. Domain structure and substrate specificity. J Biol Chem 2000;275:10983–8. CrossRef
- Gao Y, Wells L, Comer F, Parker G, Hart G. Dynamic O-glycosylation of nuclear and cytosolic proteins. Cloning and characterization of a neutral, cytosolic b-N-acetylglucosaminidase from human brain. J Biol Chem 2001;276:9838–45. CrossRef
- Roquemore E, Chou T, Hart G. As GalT labels terminal GlcNAc sugars, experiments using PNGaseF and b-elimination serve as valuable controls. Methods Enzymol 1994;230:443–60. CrossRef
- Greis K, Gibson W, Hart G. Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952. J Virol 1994;68:8339–49.
- Greis K, Hayes B, Comer F, Kirk M, Barnes S, Lowary T, Hart G. Selective detection and site-analysis of O-GlcNAc-modified glycopeptides by beta-elimination and tandem electrospray mass spectrometry. Anal Biochem 1996;234:38–49. CrossRef
- Cole RN, Hart GW. Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions. J Neurochem 1999;73:418–28. CrossRef
- Zachara NE, Gao Y, Cole RN, Hart GW. Detection and analysis of proteins modified by O-linked N-acetylglucosamine. Curr Protoc Protein Sci 2001;2:12.8.1–25.
- Zachara NE, Cheung WD, Hart GW. Nucleocytoplasmic glycosylation, O-GlcNAc: identification and site mapping. In: Dickson RC, Mendenhall MD, editors. Methods in molecular biology—signal transduction protocols. Clifton: Humana; 2003. p. 175–94.
- Whelan SA, Hart GW. Identification of O-GlcNAc sites on proteins. Methods Enzymol 2006;415:113–33. CrossRef
- Holt G, Snow C, Senior A, Haltiwanger R, Gerace L, Hart G. Nuclear pore complex glycoproteins contain cytoplasmically disposed O-linked N-acetylglucosamine. J Cell Biol 1987;104:1157–64. CrossRef
- Comer F, Vosseller K, Wells L, Accavitti MA, Hart GW. Characterization of a mouse monoclonal antibody specific for O-linked N-acetylglucosamine. Anal Biochem 2001;293:169–77. CrossRef
- Wang Z, Pandey A, Hart G. Dynamic interplay between O-Linked N-acetylglucosaminylation and glycogen synthase kinase-3-dependent phosphorylation. Mol Cell Proteomics 2007;6:1365–79. CrossRef
- Vocadlo D, Hang H, Kim E, Hanover J, Bertozzi C. A chemical approach for identifying O-GlcNAc-modified proteins in cells. Proc Natl Acad Sci U S A 2003;100:9116–21. CrossRef
- Nandi A, Sprung R, Barma D, Zhao Y, Kim S, Falck J, Zhao Y. Global identification of O-GlcNAc-modified proteins. Anal Chem 2006;78:452–8. CrossRef
- Zachara NE, Hart GW. Cell signaling, the essential role of O-GlcNAc. Biochim Biophys Acta 2006;1761:599–617.
- Nagata K, Izawa I, Inagaki M. A decade of site- and phosphorylation state-specific antibodies: recent advances in studies of spatiotemporal protein phosphorylation. Genes Cells 2001;6:653–64. CrossRef
- Haynes P, Aebersold R. Simultaneous detection and identification of O-GlcNAc-modified glycoproteins using liquid chromatography tandem mass spectrometry. Anal Chem 2000;72:5402–10. CrossRef
- Chalkley RJ, Burlingame AL. Identification of novel sites of O-Nacetylglucosamine modification of serum response factor using quadrupole time-of-flight mass spectrometry. Mol Cell Proteomics 2003;2:182–90. CrossRef
- Beausoleil S, Jedrychowski M, Schwartz D, Elias J, Villén J, Li J, Cohn M, Cantley L, Gygi S. Large-scale characterization of HeLa cell nuclear phosphoproteins. Proc Natl Acad Sci U S A 2004;101:12130–5. CrossRef
- Chi A, Huttenhower C, Geer L, Coon J, Syka J, Bai D, Schabanowitz J, Burke D, Troyanskaya O, Hunt D. Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry. Proc Natl Acad Sci U S A 2007;104:2193–8. CrossRef
- Larsen M, Thingholm T, Jensen O, Roepstoff P, Jørgensen T. Highly selective enrichment of phosphorylated peptides from peptide mixtures using titanium dioxide microcolumn. Mol Cell Proteomics 2005;4:873–86. CrossRef
- Hayes B, Greis K, Hart G. Specific isolation of O-linked N-acetylglucosamine glycopeptides from complex mixtures. Anal Biochem 1995;228:115–22. CrossRef
- Vosseller K, Trinidad J, Chalkley R, Specht C, Thalhammer A, Lynn A, Snedecor J, Guan S, Medzihradszky K, Maltby D, Schoepfer R, Burlingame A. O-Linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics 2006;5:923–34. CrossRef
- Wells L, Vosseller K, Cole R, Cronshaw J, Matunis M, Hart G. Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 2002;1:791–804. CrossRef
- McLachlin D, Chait B. Improved beta-elimination-based affinity purification strategy for enrichment of phosphopeptides. Anal Chem 2003;75:6826–36. CrossRef
- Li W, Backlund P, Boykins R, Wang G, Chen H. Susceptibility of the hydroxyl groups in serine and threonine to beta-elimination/Michael addition under commonly used moderately high-temperature conditions. Anal Biochem 2003;323:94–102. CrossRef
- Rusnak F, Zhou J, Hathaway G. Reaction of phosphorylated and O-glycosylated peptides by chemically targeted identification at ambient temperature. J Biomol Tech 2004;15:296–304.
- Ramakrishnan B, Qasba P. Structure-based design of b1,4-galactosyltransferase I (b4Gal-T1) with equally efficient N-acetylgalactosaminyltransferase activity. Point mutation broadens b4gal-T1 donor specificity. J Biol Chem 2002;277:20833–9. CrossRef
- Bülter T, Schumacher T, Namdjou D, Gutierrez Gallego R, Clausen H, Elling L. Chemoenzymatic synthesis of biotinylated nucleotide sugars as substrates for glycosyltransferases. Chembiochem 2001;2:884–94. CrossRef
- Khidekel N, Ficarro S, Peters E, Hsieh-Wilson L. Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci U S A 2004;101:13132–7. CrossRef
- Kolb H, Finn M, Sharpless K. Click chemistry: diverse chemical function from a few good reactions. Angew Chem 2001;40:2004–21. CrossRef
- Khidekel N, Ficarro S, Clark P, Bryan M, Swaney D, Rexach J, Sun Y, Coon J, Peters E, Hsieh-Wilson L. Probing the dynamics of O-GlcNAc glycosylation in the brain using quantitative proteomics. Nat Chem Biol 2007;3:339–48. CrossRef
- Zubarev R, Kelleher N, McLafferty F. ECD of multiply charged protein cations. A non-ergodic process. J Am Chem Soc 1998;120:3265–6. CrossRef
- Syka J, Coon J, Schroeder M, Shabanowitz J, Hunt DF. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci U S A 2004;101:9528–33. CrossRef
- Chi A, Bai D, Geer L, Shabanowitz J, Hunt D. Analysis of intact proteins on a chromatographic time scale by electron transfer dissociation tandem mass spectrometry. Int J Mass Spectrom 2007;259:197–203. CrossRef
- Schroeder M, Webb D, Shabanowitz J, Horwitz A, Hunt D. Methods for the detection of paxillin post-translational modifications and interacting proteins by mass spectrometry. J Proteome Res 2005;4:1832–41. CrossRef
- Dias W, Hart G. O-GlcNAc modification in diabetes and Alzheimer’s disease. Mol Biosyst 2007;3:766–72. CrossRef
- Glycomic Approaches to Study GlcNAcylation: Protein Identification, Site-mapping, and Site-specific O-GlcNAc Quantitation
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