Encyclopedia of Biophysics

Living Edition
| Editors: Gordon Roberts, Anthony Watts, European Biophysical Societies

Mass Spectrometry of N-Linked Carbohydrates and Glycoproteins

  • David John HarveyEmail author
Living reference work entry
DOI: https://doi.org/10.1007/978-3-642-35943-9_217-1


N-linked glycans are those attached to proteins with an amide bond to asparagine in an Asp-Xxx-Ser (or -Thr or, occasionally, -Cys) motif where Xxx is any amino acid except proline. Their structures are generally well defined (Stanley et al. 2017), unlike the O-linked glycans (those attached directly to serine or threonine) which, although usually smaller, have more diverse structures and are, consequently, more difficult to analyze. Typical structures of N-glycans are shown in Fig. 1. All have a common trimannosyl chitobiose (Man 3GlcNAc 2) core with several attached antennae, and their analysis, therefore, requires the identification of factors such as the type of glycan (high-mannose, hybrid, or complex (see Fig. 1)), the number and composition of each antenna, number and location of fucose residues, presence or absence of a bisecting GlcNAc residue (β1 → 4-linked to the core mannose residue), and the presence of any further substitution, particularly sialylation....
This is a preview of subscription content, log in to check access.


  1. Alley WR Jr, Mann BF, Novotny MV (2013) High-sensitivity analytical approaches for the structural characterization of glycoproteins. Chem Rev 113:2668–2732CrossRefGoogle Scholar
  2. Bitto D, Harvey DJ, Halldorsson S et al. (2015) Determination of N-linked glycosylation in UUKV glycoproteins by negative ion mass spectrometry and ion mobility. Methods Mol Biol 1331:93–121CrossRefGoogle Scholar
  3. Börnsen KO, Mohr MD, Widmer HM (1995) Ion exchange and purification of carbohydrates on a Nafion(R) membrane as a new sample pretreatment for matrix-assisted laser desorption-ionization mass spectrometry. Rapid Commun Mass Spectrom 9:1031–1034CrossRefGoogle Scholar
  4. Carpita NC, Shea EM (1989) Linkage structure of carbohydrates by gas chromatography-mass spectrometry (GC-MS) of partially methylated alditol acetates. In: Biermann CJ, McGinnis GD (eds) Analysis of carbohydrates by GLC and MS. CRC Press, Boca Raton, pp 157–216Google Scholar
  5. Carr SA, Barr JR, Roberts GD et al. (1990) Identification of attachment sites and structural classes of asparagine-linked carbohydrates in glycoproteins. Methods Enzymol 193:501–518CrossRefGoogle Scholar
  6. Ceroni A, Maass K, Geyer H et al. (2008) GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans. J Proteome Res 7:1650–1659CrossRefGoogle Scholar
  7. Domon B, Costello CE (1988) A systematic nomenclature for carbohydrate fragmentations in FAB-MS/MS spectra of glycoconjugates. Glycoconj J 5:397–409CrossRefGoogle Scholar
  8. Fenn LS, McLean JA (2009) Simultaneous glycoproteomics on the basis of structure using ion mobility-mass spectrometry. Mol BioSyst 5:1298–1302CrossRefGoogle Scholar
  9. Greer FM, Morris HR (1997) Fast-atom bombardment and electrospray mass spectrometry of peptides, proteins, and glycoproteins. Methods Mol Biol 64:147–163Google Scholar
  10. Han L, Costello CE (2013) Mass spectrometry of glycans. Biochem Mosc 78:710–720CrossRefGoogle Scholar
  11. Harvey DJ (1999) Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates. Mass Spectrom Rev 18:349–451CrossRefGoogle Scholar
  12. Harvey DJ (2009) In-gel enzymatic release of N-glycans. In: The protein protocols handbook, 3rd edn. Humana Press, New York, pp 1357–1364Google Scholar
  13. Harvey DJ (2011) Derivatization of carbohydrates for analysis by chromatography, electrophoresis and mass spectrometry. J Chromatogr B 879:1196–1225CrossRefGoogle Scholar
  14. Harvey DJ (2016a) Analysis of protein glycosylation by mass spectrometry. In: Griffiths JR, Unwin RD (eds) Analysis of protein post-translational modifications by mass spectrometry. Wiley, Hoboken, pp 89–159CrossRefGoogle Scholar
  15. Harvey DJ (2016b) Carbohydrate analysis by ESI and MALDI. In: Cole RB (ed) Electrospray and MALDI mass spectrometry fundamentals, instrumentation, practicalities and biological applications. Wiley, Hobokken, pp 723–769Google Scholar
  16. Harvey DJ (2018) Analysis of carbohydrates and glycoconjugates by matrix-assisted laser desorption/ionization mass spectrometry: an update for 2013–2014. Mass Spectrom Rev 37:353–491CrossRefGoogle Scholar
  17. Harvey DJ, Royle L, Radcliffe CM et al. (2008) Structural and quantitative analysis of N-linked glycans by MALDI and negative ion nanospray mass spectrometry. Anal Biochem 376:44–60CrossRefGoogle Scholar
  18. Harvey DJ, Merry AH, Royle L et al. (2009) Proposal for a standard system for drawing structural diagrams of N- and O-linked carbohydrates and related compounds. Proteomics 9:3796–3801CrossRefGoogle Scholar
  19. Harvey DJ, Sobott F, Crispin M et al. (2011) Ion mobility mass spectrometry for extracting spectra of N-glycans directly from incubation mixtures following glycan release: application to glycans from engineered glycoforms of intact, folded HIV gp120. J Am Soc Mass Spectrom 22:568–581CrossRefGoogle Scholar
  20. Hillenkamp F, Peter-Katalinic J (eds) (2014) MALDI MS: a practical guide to instrumentation, methods and applications. Wiley VCH, WeinheimGoogle Scholar
  21. Hossain M, Limbach PA (2010) A comparison of MALDI matrices. In: Cole RB (ed) Electrospray and MALDI mass spectrometry: fundamentals, instrumentation, practicalities and biological applications, 2nd edn. Wiley, Hoboken, pp 215–244Google Scholar
  22. Jensen PH, Karlsson NG, Kolarich D et al. (2012) Structural analysis of N- and O-glycans released from glycoproteins. Nat Protoc 7:1299–1300CrossRefGoogle Scholar
  23. Küster B, Wheeler SF, Hunter AP, Dwek RA, Harvey DJ (1997) Sequencing of N-linked oligosaccharides directly from protein gels: In-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high performance liquid chromatography. Anal Biochem 250:82–101CrossRefGoogle Scholar
  24. Mechref Y, Novotny MV, Krishnan C (2003) Structural characterization of oligosaccharides using MALDI-TOF/TOF tandem mass spectrometry. Anal Chem 75:4895–4903CrossRefGoogle Scholar
  25. Novotny MV, Alley WR Jr, Mann BF (2013) Analytical glycobiology at high sensitivity: current approaches and directions. Glycoconj J 30:89–117CrossRefGoogle Scholar
  26. Packer NH, Lawson MA, Jardine DR et al. (1998) A general approach to desalting oligosaccharides released from glycoproteins. Glycoconj J 15:737–747CrossRefGoogle Scholar
  27. Patel T, Bruce J, Merry A et al. (1993) Use of hydrazine to release in intact and unreduced form both N- and O-linked oligosaccharides from glycoproteins. Biochemistry 32:679–693CrossRefGoogle Scholar
  28. Powell AK, Harvey DJ (1996) Stabilisation of sialic acids in N-linked oligosaccharides and gangliosides for analysis by positive ion matrix-assisted laser desorption-ionization mass spectrometry. Rapid Commun Mass Spectrom 10:1027–1032CrossRefGoogle Scholar
  29. Reiding KR, Blank D, Kuijper DM et al. (2014) High-throughput profiling of protein N-glycosylation by MALDI-TOF-MS employing linkage-specific sialic acid esterification. Anal Chem 86:5784–5793CrossRefGoogle Scholar
  30. Ruhaak LR, Huhn C, Koeleman CAM et al. (2012) Robust and high-throughput sample preparation for (semi-)quantitative analysis of N-glycosylation profiles from plasma samples. Methods Mol Biol 893:371–385CrossRefGoogle Scholar
  31. Selman MHJ, Hemayatkar M, Deelder AM et al. (2011) Cotton HILIC SPE microtips for microscale purification and enrichment of glycans and glycopeptides. Anal Chem 83:2492–2499CrossRefGoogle Scholar
  32. Stanley P, Taniguchi N, Markus A (2017) Chapter 9, N-Glycans. In: Varki A, Cummings RD, Esko JD et al. (eds) Essentials of glycobiology [Internet], 3rd edn. Cold Spring Harbor Laboratory Press, New YorkGoogle Scholar
  33. Stephens E, Maslen SL, Green LG et al. (2004) Fragmentation characteristics of neutral N-linked glycans using a MALDI-TOF/TOF tandem mass spectrometer. Anal Chem 76:2343–2354CrossRefGoogle Scholar
  34. Wheeler SF, Domann P, Harvey DJ (2009) Derivatization of sialic acids for stabilization in matrix-assisted laser desorption/ionization mass spectrometry and concomitant differentiation of α(2-3) and α(2-6) isomers. Rapid Commun Mass Spectrom 23:303–312CrossRefGoogle Scholar
  35. Woodin CL, Maxon M, Desaire H (2013) Software for automated interpretation of mass spectrometry data from glycans and glycopeptides. Analyst 138:2793–2803CrossRefGoogle Scholar

Copyright information

© European Biophysical Societies' Association (EBSA) 2018

Authors and Affiliations

  1. 1.Target Discovery Institute, Nuffield Department of MedicineUniversity of OxfordOxfordUK

Section editors and affiliations

  • Neil J. Oldham
    • 1
  1. 1.School of ChemistryThe University of NottinghamNottinghamUK