N-Glycomics and N-Glycoproteomics of Human Cerebrospinal Fluid

  • Sophie Cholet
  • Arnaud Goyallon
  • Christophe Junot
  • François Fenaille
Part of the Neuromethods book series (NM, volume 127)


Protein glycosylation is one of the most complex types of posttranslational modifications of proteins. Disorders of the central nervous system can lead to particular patterns of glycans bound to proteins from cerebrospinal fluid (CSF). In this chapter, we first provide a detailed protocol for obtaining relevant mass spectrometric profiles of CSF N-glycans after permethylation (N-glycomics). In addition, we describe how to perform enrichment of tryptic glycopeptides from CSF proteins by hydrophilic interaction in an efficient and reproducible manner. Resulting glycopeptides can be either analyzed directly by nano-liquid chromatography/tandem mass spectrometry (N-glycoproteomics) or after de-N-glycosylation. We also indicate how relevant site-specific information data can be obtained for most CSF proteins by combining the data from the analysis of N-glycans, N-glycopeptides, and deglycosylated peptides.

Key words

Cerebrospinal fluid Mass spectrometry N-glycans Permethylation Glycopeptides 



This work was supported by the Commissariat à l’Energie Atomique et aux Energies Alternatives and the MetaboHUB infrastructure (ANR-11-INBS-0010 grant).


  1. 1.
    Apweiler R, Hermjakob H, Sharon N (1999) On the frequency of protein glycosylation, as deduced from analysis of the SWISS-PROT database. Biochim Biophys Acta 1473:4–8CrossRefPubMedGoogle Scholar
  2. 2.
    Ohtsubo K, Marth JD (2006) Glycosylation in cellular mechanisms of health and disease. Cell 126:855–867CrossRefPubMedGoogle Scholar
  3. 3.
    Varki A et al (2009) Essentials of glycobiology. Cold Spring Harbor Laboratory Press, Cold Spring Harbour, NYGoogle Scholar
  4. 4.
    Schachter H, Freeze HH (2009) Glycosylation diseases: quo vadis? Biochim Biophys Acta 1792:925–930CrossRefPubMedGoogle Scholar
  5. 5.
    Hennet T (2012) Diseases of glycosylation beyond classical congenital disorders of glycosylation. Biochim Biophys Acta 1820:1306–1317CrossRefPubMedGoogle Scholar
  6. 6.
    Freeze HH et al (2015) Neurological aspects of human glycosylation disorders. Annu Rev Neurosci 38:105–125CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Jaeken J (2010) Congenital disorders of glycosylation. Ann N Y Acad Sci 1214:190–198CrossRefPubMedGoogle Scholar
  8. 8.
    Lefeber DJ, Morava E, Jaeken J (2011) How to find and diagnose a CDG due to defective N-glycosylation. J Inherit Metab Dis 34:849–852CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Barone R et al (2012) Glycomics of pediatric and adulthood diseases of the central nervous system. J Proteomics 75:5123–5139CrossRefPubMedGoogle Scholar
  10. 10.
    Schedin-Weiss S, Winblad B, Tjernberg LO (2014) The role of protein glycosylation in Alzheimer disease. FEBS J 281:46–62CrossRefPubMedGoogle Scholar
  11. 11.
    Schutzer SE et al (2011) Distinct cerebrospinal fluid proteomes differentiate post-treatment lyme disease from chronic fatigue syndrome. PLoS One 6:e17287CrossRefPubMedPubMedCentralGoogle Scholar
  12. 12.
    Palmigiano A et al (2016) CSF N-glycoproteomics for early diagnosis in Alzheimer’s disease. J Proteomics 131:29–37CrossRefPubMedGoogle Scholar
  13. 13.
    Fogli A et al (2012) CSF N-glycan profiles to investigate biomarkers in brain developmental disorders: application to leukodystrophies related to eIF2B mutations. PLoS One 7:e42688CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Barone R et al (2016) CSF N-glycan profile reveals sialylation deficiency in a patient with GM2 gangliosidosis presenting as childhood disintegrative disorder. Autism Res 9:423–428CrossRefPubMedGoogle Scholar
  15. 15.
    Lefeber DJ (2016) Protein-specific glycoprofiling for patient diagnostics. Clin Chem 62:9–11CrossRefPubMedGoogle Scholar
  16. 16.
    Thaysen-Andersen M, Packer NH, Schulz BL (2016) Maturing glycoproteomics technologies provide unique structural insights into the N-glycoproteome and its regulation in health and disease. Mol Cell Proteomics 15:1773–1790CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Goyallon A et al (2015) Evaluation of a combined glycomics and glycoproteomics approach for studying the major glycoproteins present in biofluids: application to cerebrospinal fluid. Rapid Commun Mass Spectrom 29:461–473CrossRefPubMedGoogle Scholar
  18. 18.
    Ciucanu I, Kerek F (1984) A simple and rapid method for the permethylation of carbohydrates. Carbohydr Res 131:209–217CrossRefGoogle Scholar
  19. 19.
    Wada Y, Tajiri M, Yoshida S (2004) Hydrophilic affinity isolation and MALDI multiple-stage tandem mass spectrometry of glycopeptides for glycoproteomics. Anal Chem 76:6560–6565CrossRefPubMedGoogle Scholar
  20. 20.
    Wang H et al (2011) Integrated mass spectrometry-based analysis of plasma glycoproteins and their glycan modifications. Nat Protoc 6:253–269CrossRefPubMedPubMedCentralGoogle Scholar
  21. 21.
    Suckau D et al (2003) A novel MALDI LIFT-TOF/TOF mass spectrometer for proteomics. Anal Bioanal Chem 376:952–965CrossRefPubMedGoogle Scholar
  22. 22.
    Kuster B, Harvey DJ (1997) Ammonium containing buffers should be avoided during enzymatic release of glycans from glycoproteins when followed by reducing terminal derivatization. Glycobiology 7:vii–vixPubMedGoogle Scholar
  23. 23.
    Muzikar J et al (2004) Enhanced post-source decay and cross-ring fragmentation of oligosaccharides facilitated by conversion to amino derivatives. Rapid Commun Mass Spectrom 18:1513–1518CrossRefPubMedGoogle Scholar
  24. 24.
    Staubach S et al (2012) Differential glycomics of epithelial membrane glycoproteins from urinary exovesicles reveals shifts toward complex-type N-glycosylation in classical galactosemia. J Proteome Res 11:906–916CrossRefPubMedGoogle Scholar
  25. 25.
    Ceroni A et al (2008) GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 7:1650–1659CrossRefPubMedGoogle Scholar
  26. 26.
    Palmisano G et al (2012) Chemical deamidation: a common pitfall in large-scale N-linked glycoproteomic mass spectrometry-based analyses. J Proteome Res 11:1949–1957CrossRefPubMedGoogle Scholar
  27. 27.
    Fenaille F et al (2007) Site-specific N-glycan characterization of human complement factor H. Glycobiology 17:932–944CrossRefPubMedGoogle Scholar
  28. 28.
    Parker BL et al (2011) Quantitative N-linked glycoproteomics of myocardial ischemia and reperfusion injury reveals early remodeling in the extracellular environment. Mol Cell Proteomics 10:M110.006833CrossRefPubMedPubMedCentralGoogle Scholar
  29. 29.
    Alvarez-Manilla G et al (2006) Tools for glycoproteomic analysis: size exclusion chromatography facilitates identification of tryptic glycopeptides with N-linked glycosylation sites. J Proteome Res 5:701–708CrossRefPubMedGoogle Scholar
  30. 30.
    Zielinska DF et al (2010) Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints. Cell 141:897–907CrossRefPubMedGoogle Scholar
  31. 31.
    Fang P et al (2016) In-depth mapping of the mouse brain N-glycoproteome reveals widespread N-glycosylation of diverse brain proteins. Oncotarget 7:38796–38809CrossRefPubMedPubMedCentralGoogle Scholar
  32. 32.
    Lee LY et al (2016) Toward automated N-glycopeptide identification in glycoproteomics. J Proteome Res 15:3904–3915CrossRefPubMedGoogle Scholar
  33. 33.
    Desaire H (2013) Glycopeptide analysis, recent developments and applications. Mol Cell Proteomics 12:893–901CrossRefPubMedPubMedCentralGoogle Scholar
  34. 34.
    Wuhrer M et al (2007) Glycoproteomics based on tandem mass spectrometry of glycopeptides. J Chromatogr B Analyt Technol Biomed Life Sci 849:115–128CrossRefPubMedGoogle Scholar
  35. 35.
    Hinneburg H et al (2016) The art of destruction: optimizing collision energies in quadrupole-time of flight (Q-TOF) instruments for glycopeptide-based glycoproteomics. J Am Soc Mass Spectrom 27:507–519CrossRefPubMedPubMedCentralGoogle Scholar
  36. 36.
    Chen R et al (2014) Site-specific characterization of cell membrane N-glycosylation with integrated hydrophilic interaction chromatography solid phase extraction and LC-MS/MS. J Proteomics 103:194–203CrossRefPubMedGoogle Scholar
  37. 37.
    Nilsson J et al (2009) Enrichment of glycopeptides for glycan structure and attachment site identification. Nat Methods 6:809–811CrossRefPubMedGoogle Scholar

Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Sophie Cholet
    • 1
  • Arnaud Goyallon
    • 1
  • Christophe Junot
    • 1
  • François Fenaille
    • 1
  1. 1.CEA, INRA, IBITECS, Service de Pharmacologie et d’Immunoanalyse, UMR 0496, Laboratoire d’Etude du Métabolisme des Médicaments, MetaboHUB-ParisUniversité Paris SaclayGif-sur-Yvette cedexFrance

Personalised recommendations