N-Glycosylation Analysis Using the StrOligo Algorithm

  • Martin Ethier
  • Daniel Figeys
  • Hélène Perreault
Protocol
Part of the Methods in Molecular Biology™ book series (MIMB, volume 328)

Abstract

N-glycosylation of proteins is the predominant glycosylation in mammals and confers specific conformations, localization, and functions to proteins. High-throughput proteomics techniques have focused on the identification of proteins through amino acid sequence determination, with little attention paid to their post-translational modification, in particular, glycosylation. High-throughput mass spectrometric data often contain information about glycosylation, but this is systematically discarded by proteomic search engines. We have developed an algorithm, StrOligo (for STRucture of OLIGOsaccharides), capable of automated analysis of oligosaccharide composition and possible structures by mass spectrometry.

The algorithm analyzes tandem mass spectrometry (MS/MS) data in an automated three-step process and provides possible structures and a discrimination score. In the first step, the algorithm constructs a relationship tree of the monosaccharide moiety losses observed in the MS/MS spectrum. In the second step, the algorithm uses the tree to propose possible compositions and structures from combinations of adduct and fragment ions as well as a discrimination score, which reflects the fit with the experimental results. Finally, an interface is available to visualize the proposed structures and their scores. As well, the MS/MS spectrum is displayed with relevant peaks labeled for the proposed structure with the highest discrimination score, using a modified nomenclature.

Key Words

N-linked oligosaccharides mammalian cells structure determination tandem mass spectrometry automated interpretation algorithm sugar composition 

References

  1. 1.
    Viseux N., Costello C. E., and Domon B. J. (1999) Post-source decay mass spectrometry: optimized calibration procedure and structural characterization of permethylated oligosaccharides. J. Mass Spectrom. 34, 364–376.PubMedCrossRefGoogle Scholar
  2. 2.
    Harvey D. J., Bateman R. H., Bordoli R. S., and Tyldesley R. (2000) Ionisation and fragmentation of complex glycans with a quadrupole time-of-flight mass spectrometer fitted with a matrix-assisted laser desorption/ionisation ion source. Rapid Commun. Mass Spectrom. 14, 2135–2142.PubMedCrossRefGoogle Scholar
  3. 3.
    Saba J. A., Kunkel J. P., Jan D. C. H., et al. (2002) A study of immunoglobulin G glycosylation in monoclonal and polyclonal species by electrospray and matrixassisted laser desorption/ionization mass spectrometry. Anal. Biochem. 305, 16–31.PubMedCrossRefGoogle Scholar
  4. 4.
    Varki A., Cummings R., Esko J., Freeze H., Hart G., and Marth J. (eds) (1999) Essentials of Glycobiology. Cold Harbor Laboratory, Cold Spring Harbor, NY.Google Scholar
  5. 5.
    Mizuno Y., Sasagawa T., Dohmae N., and Takio K. (1999) An automated interpretation of MALDI/TOF postsource decay spectra of oligosaccharides. 1. Automated peak assignment. Anal. Chem. 71, 4764–4771.PubMedCrossRefGoogle Scholar
  6. 6.
    Cooper C. A., Gasteiger E., and Packer N. H. (2001) GlycoMod-a software tool for determining glycosylation compositions from mass spectrometric data. Proteomics 1, 340–349.PubMedCrossRefGoogle Scholar
  7. 7.
    Gaucher S. P., Morrow J., and Leary J. A. (2000) STAT: a saccharide topology analysis tool used in combination with tandem mass spectrometry. Anal. Chem. 72, 2331–2336.PubMedCrossRefGoogle Scholar
  8. 8.
    Ethier M., Saba J. A., Ens W., Standing K. G., and Perreault H. (2002) Automated structural assignment of derivatized complex N-Linked oligosaccharides from tandem mass spectra. Rapid Commun. Mass Spectrom. 16, 1743–1754.PubMedCrossRefGoogle Scholar
  9. 9.
    Ethier M., Saba J. A., Spearman M., et al. (2003) Application of the StrOligo algorithm for the automated structure assignment of complex N-Linked glycans from glycoproteins using tandem mass spectrometry. Rapid Commun. Mass Spectrom. 17, 2713–2720.PubMedCrossRefGoogle Scholar
  10. 10.
    Ethier M., Krokhin O., Ens W., Standing K. G., Wilkins J., and Perreault H. (2005) Global and site-specific detection of human integrin alpha 5 beta 1 glycosylation using tandem mass spectrometry and the StrOligo algorithm. Rapid Commun. Mass Spectrom. 19, 721–727.PubMedCrossRefGoogle Scholar
  11. 11.
    Loboda A. V., Krutchinsky A. N., Bromirski M. P., Ens W., and Standing K. G. (2000) A tandem quadrupole/time-of-flight mass spectrometer with a matrixassisted laser desorption/ionization source: design and performance. Rapid Commun. Mass Spectrom. 14, 1047–1057.PubMedCrossRefGoogle Scholar
  12. 12.
    Biemann K. and Martin S. A. (1987) Mass spectrometric determination of the amino acid sequence of peptides and proteins. Mass Spectrom. Rev. 6, 1–75.CrossRefGoogle Scholar

Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Martin Ethier
  • Daniel Figeys
  • Hélène Perreault

There are no affiliations available

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