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High-Throughput Glycomics and Glycoproteomics pp 83–96Cite as

Analysis of Permethylated Glycan by Liquid Chromatography (LC) and Mass Spectrometry (MS)

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1503))

Abstract

The development of a reliable and high-throughput glycomic profiling strategy is in high demand due to the biological roles of glycans and their association with different diseases. Native analysis can be quite difficult because of the low ionization efficiency and microheterogeneity of glycans. In this chapter, the sample preparation protocols and LC-MS analysis of permethylated glycan strategies are introduced. Solid-phase permethylation is a fast, convenient, and high-yield method to stabilize sialic acid and improve glycan ionization efficiency and analysis in positive mode; this results in a more sensitive and reliable glycomic profiling strategy. Several modifications in the LC method are also mentioned in this chapter. Online purification simplifies sample preparation and reduces sample loss. Elevating the column temperature significantly improves the peak shape of permethylated glycans and results in isomeric separation. The identification and quantification of permethylated glycans can be achieved through high resolution MS and MS/MS experiments using a MRM method; both approaches are reliable, sensitive, and conducive to high-throughput glycomic studies.

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References

  1. Haslam SM, North SJ, Dell A (2006) Mass spectrometric analysis of N- and O-glycosylation of tissues and cells. Curr Opin Struct Biol 16(5):584–591

    Article  CAS  PubMed  Google Scholar 

  2. Raju TS (2008) Terminal sugars of Fc glycans influence antibody effector functions of IgGs. Curr Opin Immunol 20(4):471–478

    Article  CAS  PubMed  Google Scholar 

  3. Back NK et al. (1994) An N-glycan within the human immunodeficiency virus type 1 gp120 V3 loop affects virus neutralization. Virology 199(2):431–438

    Article  CAS  PubMed  Google Scholar 

  4. Kolchinsky P et al. (2001) Loss of a single N-linked glycan allows CD4-independent human immunodeficiency virus type 1 infection by altering the position of the gp120 V1/V2 variable loops. J Virol 75(7):3435–3443

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  5. Doores KJ et al. (2010) Envelope glycans of immunodeficiency virions are almost entirely oligomannose antigens. Proc Natl Acad Sci U S A 107(31):13800–13805

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  6. Dennis JW, Granovsky M, Warren CE (1999) Protein glycosylation in development and disease. Bioassays 21(5):412–421

    Article  CAS  Google Scholar 

  7. Lowe JB, Marth JD (2003) A genetic approach to mammalian glycan function. Annu Rev Biochem 72:643–691

    Article  CAS  PubMed  Google Scholar 

  8. Mechref Y et al. (2012) Identifying cancer biomarkers by mass spectrometry-based glycomics. Electrophoresis 33(12):1755–1767

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  9. Ciucanu I, Kerek F (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res 131(2):209–217

    Article  CAS  Google Scholar 

  10. Ciucanu I, Costello CE (2003) Elimination of oxidative degradation during the per-O-methylation of carbohydrates. J Am Chem Soc 125(52):16213–16219

    Article  CAS  PubMed  Google Scholar 

  11. Kang P et al. (2005) Solid-phase permethylation of glycans for mass spectrometric analysis. Rapid Commun Mass Spectrom 19(23):3421–3428

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  12. Hu Y, Mechref Y (2012) Comparing MALDI-MS, RP-LC-MALDI-MS and RP-LC-ESI-MS glycomic profiles of permethylated N-glycans derived from model glycoproteins and human blood serum. Electrophoresis 33(12):1768–1777

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  13. Desantos-Garcia JL et al. (2011) Enhanced sensitivity of LC-MS analysis of permethylated N-glycans through online purification. Electrophoresis 32(24):3516–3525

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  14. Zhou S, Hu Y, Mechref Y High-temperature LC-MS analysis of permethylated glycans derived from glycoproteins. Electrophoresis 37:1506–1513

    Google Scholar 

  15. Zhou S et al. (2015) Quantitation of permethylated N-glycans through multiple-reaction monitoring (MRM) LC-MS/MS. J Am Soc Mass Spectrom 26(4):596–603

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  16. Gil GC, Velander WH, Van Cott KE (2009) N-glycosylation microheterogeneity and site occupancy of an Asn-X-Cys sequon in plasma-derived and recombinant protein C. Proteomics 9(9):2555–2567

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  17. Ahn YH et al. (2009) Quantitative analysis of an aberrant glycoform of TIMP1 from colon cancer serum by L-PHA-enrichment and SISCAPA with MRM mass spectrometry. J Proteome Res 8(9):4216–4224

    Article  CAS  PubMed  Google Scholar 

  18. Kurogochi M et al. (2010) Sialic acid-focused quantitative mouse serum glycoproteomics by multiple reaction monitoring assay. Mol Cell Proteomics 9(11):2354–2368

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  19. Zhao Y et al. (2011) Fragmentation and site-specific quantification of core fucosylated glycoprotein by multiple reaction monitoring-mass spectrometry. Anal Chem 83(22):8802–8809

    Article  CAS  PubMed  Google Scholar 

  20. Yu CY et al. (2013) Automated annotation and quantification of glycans using liquid chromatography-mass spectrometry. Bioinformatics 29(13):1706–1707

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  21. Hu Y et al. (2015) Automated annotation and quantitation of glycans by liquid chromatography/electrospray ionization mass spectrometric analysis using the MultiGlycan-ESI computational tool. Rapid Commun Mass Spectrom 29(1):135–142

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  22. Apte A, Meitei NS (2010) Bioinformatics in glycomics: glycan characterization with mass spectrometric data using SimGlycan. Methods Mol Biol 600:269–281

    Article  CAS  PubMed  Google Scholar 

  23. Meitei NS et al. (2015) Automating mass spectrometry-based quantitative glycomics using aminoxy tandem mass tag reagents with SimGlycan. J Proteomics 127(Pt A):211–222

    Article  CAS  PubMed  Google Scholar 

  24. Ceroni A et al. (2008) GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 7(4):1650–1659

    Article  CAS  PubMed  Google Scholar 

  25. Damerell D et al. (2015) Annotation of glycomics MS and MS/MS spectra using the GlycoWorkbench software tool. Methods Mol Biol 1273:3–15

    Article  CAS  PubMed  Google Scholar 

  26. Damerell D et al. (2012) The GlycanBuilder and GlycoWorkbench glycoinformatics tools: updates and new developments. Biol Chem 393(11):1357–1362

    Article  CAS  PubMed  Google Scholar 

  27. Goldberg D et al. (2005) Automatic annotation of matrix-assisted laser desorption/ionization N-glycan spectra. Proteomics 5(4):865–875

    Article  CAS  PubMed  Google Scholar 

  28. Cooper CA, Gasteiger E, Packer NH (2001) GlycoMod—a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 1(2):340–349

    Article  CAS  PubMed  Google Scholar 

  29. Maass K et al. (2007) “Glyco-peakfinder”—de novo composition analysis of glycoconjugates. Proteomics 7(24):4435–4444

    Article  CAS  PubMed  Google Scholar 

  30. Vakhrushev SY, Dadimov D, Peter-Katalinic J (2009) Software platform for high-throughput glycomics. Anal Chem 81(9):3252–3260

    Article  CAS  PubMed  Google Scholar 

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Authors and Affiliations

  1. Department of Chemistry and Biochemistry, Texas Tech University, Lubbock, TX, 79409-1061, USA

    Shiyue Zhou, Kerry M. Wooding & Yehia Mechref

Authors
  1. Shiyue Zhou
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  2. Kerry M. Wooding
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  3. Yehia Mechref
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Corresponding author

Correspondence to Yehia Mechref .

Editor information

Editors and Affiliations

  1. Faculty of Pharmacy and Biochemistry, University of Zagreb, Zagreb, Croatia

    Gordan Lauc

  2. Center for Proteomics and Metabolomics, Leiden University Medical Center, Leiden, The Netherlands

    Manfred Wuhrer

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Zhou, S., Wooding, K.M., Mechref, Y. (2017). Analysis of Permethylated Glycan by Liquid Chromatography (LC) and Mass Spectrometry (MS). In: Lauc, G., Wuhrer, M. (eds) High-Throughput Glycomics and Glycoproteomics. Methods in Molecular Biology, vol 1503. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6493-2_7

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  • DOI: https://doi.org/10.1007/978-1-4939-6493-2_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6491-8

  • Online ISBN: 978-1-4939-6493-2

  • eBook Packages: Springer Protocols

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