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Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography

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Mass Spectrometry of Glycoproteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2271))

Abstract

Glycosylation is a biologically important and complex protein posttranslational modification. The emergence of glycoproteomic technologies to identify and characterize glycans on proteins has the potential to enable a better understanding the role of glycosylation in biology, disease states, and other areas of interest. In particular, the analysis of intact glycopeptides by mass spectrometry allows information about glycan location and composition to be ascertained. However, such analysis is often complicated by extensive glycan diversity and the low abundance of glycopeptides in a complex mixture relative to nonglycosylated peptides. Enrichment of glycopeptides from a protein enzymatic digest is an effective approach to overcome such challenges. In this chapter, we described a glycopeptide enrichment method combining strong anion exchange, electrostatic repulsion, and hydrophilic interaction chromatography (SAX-ERLIC). Following enzymatic digestion of proteins into peptides, SAX-ERLIC is performed by solid phase extraction to enrich glycopeptides from biological samples with subsequent LC-MS/MS analysis. Glycopeptide data generated using the SAX-ERLIC enrichment yields a high number of total and unique glycopeptide identifications which can be mapped back to proteins. The enrichment strategy is robust, easy to perform, and does not require cleavage of glycans prior to LC-MS/MS analysis.

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References

  1. Varki A, Cummings RD et al (eds) (2015) Essentials of glycobiology. Cold Spring Harbor (NY), New York

    Google Scholar 

  2. Reily C, Stewart TJ, Renfrow MB, Novak J (2019) Glycosylation in health and disease. Nat Rev Nephrol 15(6):346–366. https://doi.org/10.1038/s41581-019-0129-4

    Article  PubMed  PubMed Central  Google Scholar 

  3. Zhu J, Sun Z, Cheng K, Chen R, Ye M, Xu B, Sun D, Wang L, Liu J, Wang F, Zou H (2014) Comprehensive mapping of protein N-glycosylation in human liver by combining hydrophilic interaction chromatography and hydrazide chemistry. J Proteome Res 13(3):1713–1721. https://doi.org/10.1021/pr401200h

    Article  CAS  PubMed  Google Scholar 

  4. Zhang H, Li XJ, Martin DB, Aebersold R (2003) Identification and quantification of N-linked glycoproteins using hydrazide chemistry, stable isotope labeling and mass spectrometry. Nat Biotechnol 21(6):660–666. https://doi.org/10.1038/nbt827

    Article  CAS  PubMed  Google Scholar 

  5. Liu T, Qian WJ, Gritsenko MA, Camp DG 2nd, Monroe ME, Moore RJ, Smith RD (2005) Human plasma N-glycoproteome analysis by immunoaffinity subtraction, hydrazide chemistry, and mass spectrometry. J Proteome Res 4(6):2070–2080. https://doi.org/10.1021/pr0502065

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Li X, Jiang J, Zhao X, Wang J, Han H, Zhao Y, Peng B, Zhong R, Ying W, Qian X (2013) N-glycoproteome analysis of the secretome of human metastatic hepatocellular carcinoma cell lines combining hydrazide chemistry, HILIC enrichment and mass spectrometry. PLoS One 8(12):e81921. https://doi.org/10.1371/journal.pone.0081921

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  7. Magdeldin S, Yamamoto K, Yoshida Y, Xu B, Zhang Y, Fujinaka H, Yaoita E, Yates JR 3rd, Yamamoto T (2014) Deep proteome mapping of mouse kidney based on OFFGel prefractionation reveals remarkable protein post- translational modifications. J Proteome Res 13(3):1636–1646. https://doi.org/10.1021/pr401122m

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  8. Zhang B, Sheng Q, Li X, Liang Q, Yan J, Liang X (2011) Selective enrichment of glycopeptides for mass spectrometry analysis using C18 fractionation and titanium dioxide chromatography. J Sep Sci 34(19):2745–2750. https://doi.org/10.1002/jssc.201100427

    Article  CAS  PubMed  Google Scholar 

  9. Zhu J, Wang F, Cheng K, Dong J, Sun D, Chen R, Wang L, Ye M, Zou H (2013) A simple integrated system for rapid analysis of sialic-acid-containing N-glycopeptides from human serum. Proteomics 13(8):1306–1313. https://doi.org/10.1002/pmic.201200367

    Article  CAS  PubMed  Google Scholar 

  10. Palmisano G, Parker BL, Engholm-Keller K, Lendal SE, Kulej K, Schulz M, Schwammle V, Graham ME, Saxtorph H, Cordwell SJ, Larsen MR (2012) A novel method for the simultaneous enrichment, identification, and quantification of phosphopeptides and sialylated glycopeptides applied to a temporal profile of mouse brain development. Mol Cell Proteomics 11(11):1191–1202. https://doi.org/10.1074/mcp.M112.017509

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  11. Palmisano G, Lendal SE, Larsen MR (2011) Titanium dioxide enrichment of sialic acid-containing glycopeptides. Methods Mol Biol 753:309–322. https://doi.org/10.1007/978-1-61779-148-2_21

    Article  CAS  PubMed  Google Scholar 

  12. Larsen MR, Jensen SS, Jakobsen LA, Heegaard NH (2007) Exploring the sialiome using titanium dioxide chromatography and mass spectrometry. Mol Cell Proteomics 6(10):1778–1787. https://doi.org/10.1074/mcp.M700086-MCP200

    Article  CAS  PubMed  Google Scholar 

  13. Kirwan A, Utratna M, O’Dwyer ME, Joshi L, Kilcoyne M (2015) Glycosylation-based serum biomarkers for cancer diagnostics and prognostics. Biomed Res Int 2015:490531. https://doi.org/10.1155/2015/490531

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Wuhrer M, Catalina MI, Deelder AM, Hokke CH (2007) Glycoproteomics based on tandem mass spectrometry of glycopeptides. J Chromatogr B Analyt Technol Biomed Life Sci 849(1-2):115–128. https://doi.org/10.1016/j.jchromb.2006.09.041

    Article  CAS  PubMed  Google Scholar 

  15. Huang BY, Yang CK, Liu CP, Liu CY (2014) Stationary phases for the enrichment of glycoproteins and glycopeptides. Electrophoresis 35(15):2091–2107. https://doi.org/10.1002/elps.201400034

    Article  CAS  PubMed  Google Scholar 

  16. Chen CC, Su WC, Huang BY, Chen YJ, Tai HC, Obena RP (2014) Interaction modes and approaches to glycopeptide and glycoprotein enrichment. Analyst 139(4):688–704. https://doi.org/10.1039/c3an01813j

    Article  CAS  PubMed  Google Scholar 

  17. Ongay S, Boichenko A, Govorukhina N, Bischoff R (2012) Glycopeptide enrichment and separation for protein glycosylation analysis. J Sep Sci 35(18):2341–2372. https://doi.org/10.1002/jssc.201200434

    Article  CAS  PubMed  Google Scholar 

  18. Lis H, Sharon N (1993) Protein glycosylation. Structural and functional aspects. Eur J Biochem 218(1):1–27

    Article  CAS  Google Scholar 

  19. Varki A, Cummings RD, Esko JD, Freeze HH, Stanley P, Bertozzi CR, Hart GW, Etzler ME (2009) Essentials of glycobiology. Essentials of glycobiology, 2nd edn. Cold Spring Harbor Laboratory Press, Cold Spring Harbor (NY), New York

    Google Scholar 

  20. 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(45):42530–42539. https://doi.org/10.1074/jbc.M206114200

    Article  CAS  PubMed  Google Scholar 

  21. Qiu R, Regnier FE (2005) Use of multidimensional lectin affinity chromatography in differential glycoproteomics. Anal Chem 77(9):2802–2809. https://doi.org/10.1021/ac048751x

    Article  CAS  PubMed  Google Scholar 

  22. Geng M, Zhang X, Bina M, Regnier F (2001) Proteomics of glycoproteins based on affinity selection of glycopeptides from tryptic digests. J Chromatogr B Biomed Sci Appl 752(2):293–306

    Article  CAS  Google Scholar 

  23. Alvarez-Manilla G, Warren NL, Atwood J 3rd, Orlando R, Dalton S, Pierce M (2010) Glycoproteomic analysis of embryonic stem cells: identification of potential glycobiomarkers using lectin affinity chromatography of glycopeptides. J Proteome Res 9(5):2062–2075. https://doi.org/10.1021/pr8007489

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  24. Picariello G, Ferranti P, Mamone G, Roepstorff P, Addeo F (2008) Identification of N-linked glycoproteins in human milk by hydrophilic interaction liquid chromatography and mass spectrometry. Proteomics 8(18):3833–3847. https://doi.org/10.1002/pmic.200701057

    Article  CAS  PubMed  Google Scholar 

  25. Neue K, Mormann M, Peter-Katalinic J, Pohlentz G (2011) Elucidation of glycoprotein structures by unspecific proteolysis and direct nanoESI mass spectrometric analysis of ZIC-HILIC-enriched glycopeptides. J Proteome Res 10(5):2248–2260. https://doi.org/10.1021/pr101082c

    Article  CAS  PubMed  Google Scholar 

  26. Mysling S, Palmisano G, Hojrup P, Thaysen-Andersen M (2010) Utilizing ion-pairing hydrophilic interaction chromatography solid phase extraction for efficient glycopeptide enrichment in glycoproteomics. Anal Chem 82(13):5598–5609. https://doi.org/10.1021/ac100530w

    Article  CAS  PubMed  Google Scholar 

  27. Kondo A, Thaysen-Andersen M, Hjerno K, Jensen ON (2010) Characterization of sialylated and fucosylated glycopeptides of beta2-glycoprotein I by a combination of HILIC LC and MALDI MS/MS. J Sep Sci 33(6-7):891–902. https://doi.org/10.1002/jssc.200900802

    Article  CAS  PubMed  Google Scholar 

  28. Alpert AJ (2008) Electrostatic repulsion hydrophilic interaction chromatography for isocratic separation of charged solutes and selective isolation of phosphopeptides. Anal Chem 80(1):62–76. https://doi.org/10.1021/ac070997p

    Article  CAS  PubMed  Google Scholar 

  29. Totten SM, Feasley CL, Bermudez A, Pitteri SJ (2017) Parallel comparison of N-linked Glycopeptide enrichment techniques reveals extensive Glycoproteomic analysis of plasma enabled by SAX-ERLIC. J Proteome Res 16(3):1249–1260. https://doi.org/10.1021/acs.jproteome.6b00849

    Article  CAS  PubMed  Google Scholar 

  30. Sok Hwee Cheow E, Hwan Sim K, de Kleijn D, Neng Lee C, Sorokin V, Sze SK (2015) Simultaneous enrichment of plasma soluble and extracellular vesicular glycoproteins using prolonged ultracentrifugation-electrostatic repulsion-hydrophilic interaction chromatography (PUC-ERLIC) approach. Mol Cell Proteomics 14(6):1657–1671. https://doi.org/10.1074/mcp.O114.046391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  31. Pagel O, Loroch S, Sickmann A, Zahedi RP (2015) Current strategies and findings in clinically relevant post-translational modification-specific proteomics. Expert Rev Proteomics 12(3):235–253. https://doi.org/10.1586/14789450.2015.1042867

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  32. Kuo CW, Wu IL, Hsiao HH, Khoo KH (2012) Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles. Anal Bioanal Chem 402(9):2765–2776. https://doi.org/10.1007/s00216-012-5724-1

    Article  CAS  PubMed  Google Scholar 

  33. Hao P, Zhang H, Sze SK (2011) Application of electrostatic repulsion hydrophilic interaction chromatography to the characterization of proteome, glycoproteome, and phosphoproteome using nano LC-MS/MS. Methods Mol Biol 790:305–318. https://doi.org/10.1007/978-1-61779-319-6_23

    Article  CAS  PubMed  Google Scholar 

  34. Darula Z, Sarnyai F, Medzihradszky KF (2016) O-glycosylation sites identified from mucin core-1 type glycopeptides from human serum. Glycoconj J 33(3):435–445. https://doi.org/10.1007/s10719-015-9630-6

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  35. Cao L, Yu L, Guo Z, Li X, Xue X, Liang X (2013) Application of a strong anion exchange material in electrostatic repulsion-hydrophilic interaction chromatography for selective enrichment of glycopeptides. J Chromatogr A 1299:18–24. https://doi.org/10.1016/j.chroma.2013.05.037

    Article  CAS  PubMed  Google Scholar 

  36. 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. https://doi.org/10.1155/2012/560391

    Article  CAS  PubMed  PubMed Central  Google Scholar 

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Authors and Affiliations

  1. Canary Center at Stanford for Cancer Early Detection, Department of Radiology, Stanford University School of Medicine, Palo Alto, CA, USA

    Abel Bermudez & Sharon J. Pitteri

Authors
  1. Abel Bermudez
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  2. Sharon J. Pitteri
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Correspondence to Sharon J. Pitteri .

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  1. Quality Assistance S.A., Donstiennes, Belgium

    Arnaud Delobel

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Bermudez, A., Pitteri, S.J. (2021). Enrichment of Intact Glycopeptides Using Strong Anion Exchange and Electrostatic Repulsion Hydrophilic Interaction Chromatography. In: Delobel, A. (eds) Mass Spectrometry of Glycoproteins. Methods in Molecular Biology, vol 2271. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1241-5_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1241-5_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1240-8

  • Online ISBN: 978-1-0716-1241-5

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