Abstract
In this chapter we describe the application of lectin weak affinity chromatography (LWAC) for the enrichment of peptides modified by O-linked β-N-acetylglucosamine (O-GlcNAc). O-GlcNAc is a single carbohydrate moiety post-translational modification of intracellular proteins. The stoichiometry of the modification is low and identification of the sites of O-GlcNAc attachment is challenging. To map O-GlcNAc sites we use the approach where a protein sample of interest is digested with trypsin and subjected to LWAC, which employs weak interaction between lectin wheat germ agglutinin and O-GlcNAc. Obtained sample is enriched with O-GlcNAc-modified peptides, which can be identified by means of mass spectrometry.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Hart GW (1997) Dynamic O-linked glycosylation of nuclear and cytoskeletal proteins. Annu Rev Biochem 66:315–335
Wells L, Vosseller K, Hart GW (2001) Glycosylation of nucleocytoplasmic proteins: signal transduction and O-GlcNAc. Science 291:2376–2378
Hart GW, Housley MP, Slawson C (2007) Cycling of O-linked beta-N-acetylglucosamine on nucleocytoplasmic proteins. Nature 446:1017–1022
Watson LJ, Facundo HT, Ngoh GA, Ameen M, Brainard RE, Lemma KM, Long BW, Prabhu SD, Xuan YT, Jones SP (2010) O-linked β-N-acetylglucosamine transferase is indispensable in the failing heart. Proc Natl Acad Sci USA 107:17797–17802
Cole RN, Hart GW (1999) Glycosylation sites flank phosphorylation sites on synapsin I: O-linked N-acetylglucosamine residues are localized within domains mediating synapsin I interactions. J Neurochem 73:418–428
Greis KD, Gibson W, Hart GW (1994) Site-specific glycosylation of the human cytomegalovirus tegument basic phosphoprotein (UL32) at serine 921 and serine 952. J Virol 68:8339–8349
Khidekel N, Ficarro SB, Peters EC, Hsieh-Wilson LC (2004) Exploring the O-GlcNAc proteome: direct identification of O-GlcNAc-modified proteins from the brain. Proc Natl Acad Sci USA 101:13132–13137
Nandi A, Sprung R, Barma DK, Zhao Y, Kim SC, Falck JR (2006) Global identification of O-GlcNAc-modified proteins. Anal Chem 78:452–458
Vosseller K, Hansen KC, Chalkley RJ, Trinidad JC, Wells L, Hart GW, Burlingame AL (2005) Quantitative analysis of both protein expression and serine/threonine post-translational modifications through stable isotope labeling with dithiothreitol. Proteomics 5:388–398
Wells L, Vosseller K, Cole RN, Cronshaw JM, Matunis MJ, Hart GW (2002) Mapping sites of O-GlcNAc modification using affinity tags for serine and threonine post-translational modifications. Mol Cell Proteomics 1:791–804
Chalkley RJ, Thalhammer A, Schoepfer R, Burlingame AL (2009) Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides. Proc Natl Acad Sci USA 106:8894–8899
Bhavanandan VP, Katlic AW (1979) The interaction of wheat germ agglutinin with sialoglycoproteins. The role of sialic acid. J Biol Chem 254:4000–4008
Gallagher JT, Morris A, Dexter TM (1985) Identification of two binding sites for wheat-germ agglutinin on polylactosamine-type oligosaccharides. Biochem J 231:115–122
Neumann D, Kohlbacher O, Lenhof HP, Lehr CM (2002) Lectin-sugar interaction. Calculated versus experimental binding energies. Eur J Biochem 269:1518–1524
Ohlson S, Bergstrom M, Leickt L, Zopf D (1998) Weak affinity chromatography of small saccharides with immobilised wheat germ agglutinin and its application to monitoring of carbohydrate transferase activity. Bioseparation 7:101–105
Vosseller K, Trinidad JC, Chalkley RJ, Specht CG, Thalhammer A, Lynn AJ, Snedecor JO, Guan S, Medzihradszky KF, Maltby DA, Schoepfer R, Burlingame AL (2006) O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry. Mol Cell Proteomics 5:923–934
Skorobogatko YV, Deuso J, Adolf-Bergfoyle J, Nowak MG, Gong Y, Lippa CF, Vosseller K (2010) Human Alzheimer’s disease synaptic O-GlcNAc site mapping and iTRAQ expression proteomics with ion trap mass spectrometry. Amino Acids 40:765–779
Dauphinee SM, Ma M, Too CK (2005) Role of O-linked beta-N-acetylglucosamine modification in the subcellular distribution of alpha4 phosphoprotein and Sp1 in rat lymphoma cells. J Cell Biochem 96:579–588
Kwak TK, Kim H, Jung O, Lee SA, Kang M, Kim HJ, Park JM, Kim SH, Lee JW (2010) Glucosamine treatment-mediated O-GlcNAc modification of paxillin depends on adhesion state of rat insulinoma INS-1 cells. J Biol Chem 285:36021–36031
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Ma, Z.Y., Skorobogatko, Y., Vosseller, K. (2013). Tandem Lectin Weak Affinity Chromatography for Glycoprotein Enrichment. In: Kohler, J., Patrie, S. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 951. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-146-2_2
Download citation
DOI: https://doi.org/10.1007/978-1-62703-146-2_2
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-145-5
Online ISBN: 978-1-62703-146-2
eBook Packages: Springer Protocols