Abstract
Large-scale analysis of protein N- and O-linked glycosylation by mass spectrometry has traditionally been performed in eukaryotes by parallel approaches aimed at elucidating glycan structures (glycomics) and their formerly glycosylated peptides (glycoproteomics) without reference to their intact state. Such analyses depend heavily on commercial glycosidases (e.g. protein N-glycosidase F) that can remove glycans from the peptide backbone for separate analyses. Bacterial glycosylation has only recently been identified as a widespread phenomenon. In many cases however, unique bacterial sugars preclude enzymatic removal, therefore ultimately requiring a site-specific approach for intact glycopeptide analysis. Here, we describe protocols for the enrichment of bacterial glycopeptides using zwitterionic–hydrophilic interaction liquid chromatography (ZIC-HILIC) and their analysis using liquid chromatography coupled to electrospray ionization tandem mass spectrometry (LC-MS/MS) with collision-induced dissociation (CID) and higher energy collisional dissociation (HCD) fragmentation for glycan structure elucidation and glycopeptide identification.
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References
Iwashkiw JA, Vozza NF, Kinsella RL, Feldman MF (2013) Pour some sugar on it: the expanding world of bacterial protein O-linked glycosylation. Mol Microbiol 89:14–28
Nothaft H, Szymanski CM (2010) Protein glycosylation in bacteria: sweeter than ever. Nat Rev Microbiol 8:765–778
Szymanski CM, Burr DH, Guerry P (2002) Campylobacter protein glycosylation affects host cell interactions. Infect Immun 70: 2242–2244
Howard SL, Jagannathan A, Soo EC, Hui JP, Aubry AJ, Ahmed I, Karlyshev A, Kelly JF, Jones MA, Stevens MP, Logan SM, Wren BW (2009) Campylobacter jejuni glycosylation island important in cell charge, legionaminic acid biosynthesis, and colonization of chickens. Infect Immun 77:2544–2556
Iwashkiw JA, Seper A, Weber BS, Scott NE, Vinogradov E, Stratilo C, Reiz B, Cordwell SJ, Whittal R, Schild S, Feldman MF (2012) Identification of a general O-linked protein glycosylation system in Acinetobacter baumannii and its role in virulence and biofilm formation. PLoS Pathog 8:e1002758
Lithgow KV, Scott NE, Iwashkiw JA, Thomson ELS, Foster LJ, Feldman MF, Dennis JJ (2014) A general protein O-glycosylation system within the Burkholderia cepacia complex is involved in motility and virulence. Mol Microbiol 92:116–137
Zielinska DF, Gnad F, Wisniewski JR, Mann M (2010) Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 141:897–907
Zhang H, Li X, Martin DB, Aebersold R (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21:660–666
Fredriksen L, Moen A, Adzhubei AA, Mathiesen G, Eijsink VG, Egge-Jacobsen W (2013) Lactobacillus plantarum WCFS1 O-linked protein glycosylation: an extended spectrum of target proteins and modification sites detected by mass spectrometry. Glycobiology 23:1439–1451
Nothaft H, Scott NE, Vinogradov E, Liu X, Hu R, Beadle B, Fodor C, Miller WG, Li J, Cordwell SJ, Szymanski CM (2012) Diversity in the protein N-glycosylation pathways within the Campylobacter genus. Mol Cell Proteomics 11:1203–1219
Thibault P, Logan SM, Kelly JF, Brisson JR, Ewing CP, Trust TJ, Guerry P (2001) Identification of the carbohydrate moieties and glycosylation motifs in Campylobacter jejuni flagellin. J Biol Chem 276:34862–34870
Grass S, Lichti CF, Townsend RR, Gross J, St Geme JW 3rd (2010) The Haemophilus influenzae HMW1C protein is a glycosyltransferase that transfers hexose residues to asparagine sites in the HMW1 adhesin. PLoS Pathog 6:e1000919
Verma A, Schirm M, Arora SK, Thibault P, Logan SM, Ramphal R (2006) Glycosylation of b-Type flagellin of Pseudomonas aeruginosa: structural and genetic basis. J Bacteriol 188: 4395–4403
Scott NE, Nothaft H, Edwards AV, Labbate M, Djordjevic SP, Larsen MR, Szymanski CM, Cordwell SJ (2012) Modification of the Campylobacter jejuni N-linked glycan by EptC-mediated addition of phosphoethanolamine. J Biol Chem 287:29384–29396
Twine SM, Reid CW, Aubry A, McMullin DR, Fulton KM, Austin J, Logan SM (2009) Motility and flagellar glycosylation in Clostridium difficile. J Bacteriol 191:7050–7062
Gault J, Malosse C, Dumenil G, Chamot-Rooke J (2013) A combined mass spectrometry strategy for complete posttranslational modification mapping of Neisseria meningitidis major pilin. J Mass Spectrom 48:1199–1206
Young NM, Brisson JR, Kelly J, Watson DC, Tessier L, Lanthier PH, Jarrell HC, Cadotte N, St Michael F, Aberg E, Szymanski CM (2002) Structure of the N-linked glycan present on multiple glycoproteins in the Gram-negative bacterium, Campylobacter jejuni. J Biol Chem 277:42530–42539
Scott NE, Parker BL, Connolly AM, Paulech J, Edwards AV, Crossett B, Falconer L, Kolarich D, Djordjevic SP, Højrup P, Packer NH, Larsen MR, Cordwell SJ (2011) Simultaneous glycan-peptide characterization using hydrophilic interaction chromatography and parallel fragmentation by CID, higher energy collisional dissociation, and electron transfer dissociation MS applied to the N-linked glycoproteome of Campylobacter jejuni. Mol Cell Proteomics 10:M000031–MCP000201
Scott NE, Kinsella RL, Edwards AV, Larsen MR, Dutta SM, Saba J, Foster LJ, Feldman MF (2014) Diversity within the O-linked protein glycosylation systems of Acinetobacter species. Mol Cell Proteomics 13(9):2354–2370
Rappsilber J, Mann M, Ishihama Y (2007) Protocol for micro-purification, enrichment, pre-fractionation and storage of peptides for proteomics using StageTips. Nat Protoc 2:1896–1906
Kollipara L, Zahedi RP (2013) Protein carbamylation: in vivo modification or in vitro artefact? Proteomics 13:941–944
Gobom J, Nordhoff E, Mirgorodskaya E, Ekman R, Roepstorff P (1999) Sample purification and preparation technique based on nano-scale reversed-phase columns for the sensitive analysis of complex peptide mixtures by matrix-assisted laser desorption/ionization mass spectrometry. J Mass Spectrom 34:105–116
Mysling S, Palmisano G, Højrup P, Thaysen-Andersen M (2010) Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics. Anal Chem 82:5598–5609
Larsen MR, Hojrup P, Roepstorff P (2005) Characterization of gel-separated glycoproteins using two-step proteolytic digestion combined with sequential microcolumns and mass spectrometry. Mol Cell Proteomics 4:107–119
Wu SW, Pu TH, Viner R, Khoo KH (2014) Novel LC-MS(2) product dependent parallel data acquisition function and data analysis workflow for sequencing and identification of intact glycopeptides. Anal Chem 86:5478–5486
Saba J, Dutta S, Hemenway E, Viner R (2012) Increasing the productivity of glycopeptides analysis by using higher-energy collision dissociation-accurate mass-product-dependent electron transfer dissociation. Int J Proteomics 2012:560391
Acknowledgments
This work was supported by an Australian Research Council Discovery Project Grant to S.J.C. (ARC DP 110103573). N.E.S. is supported by a National Health and Medical Research Council of Australia (NHMRC) Overseas (Biomedical) Early Career Fellowship (APP1037373) and a Michael Smith Foundation for Health Research Trainee Postdoctoral Fellowship (award # 5363).
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Scott, N.E., Cordwell, S.J. (2015). Enrichment and Identification of Bacterial Glycopeptides by Mass Spectrometry. In: Posch, A. (eds) Proteomic Profiling. Methods in Molecular Biology, vol 1295. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2550-6_25
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DOI: https://doi.org/10.1007/978-1-4939-2550-6_25
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