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Mass Spectrometry of Proteins and Peptides pp 161–180Cite as

Assigning Glycosylation Sites and Microheterogeneities in Glycoproteins by Liquid Chromatography/Tandem Mass Spectrometry

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Part of the book series: Methods In Molecular Biology ((MIMB,volume 492))

Summary

Glycosylation of proteins is one of the most common posttranslational modifications which has its bearing on function and biological activity. Assigning the glycosylation sites and their inherent microheterogenei-ties are key structural issues addressing various glycoprotein functions. This chapter describes three different approaches all based on liquid chromatography/tandem mass spectrometry (LC/MS-MS), which are commonly employed for the assignment of protein glycosylation sites and their microheterogeneities. Comparing the LC/MS-MS analysis of a native glycoprotein tryptic digest to that of a deglycosylated tryptic digest can be accomplished through a routine LC/MS instrument. The use of a scanning mass spectrometer capable of switching between high-voltage and low-voltage scans, combined with monitoring carbohydrate-characteristic oxonium ions, is yet another analytical approach utilized for characterization of the glycosylation sites of glycoproteins. These two approaches do not address the problem originating from the ion suppression associated with coeluting peptides. The use of on-line glycopeptide enrichment in conjunction with LC/MS-MS is a third approach, which reduces ion suppression, thus offering a more sensitive approach to the characterization of protein glycosylation sites.

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Acknowledgments

This work was supported by grants No. GM24349 from the National Institutes of Health (NIH) and No. RR018942 from NCRR/NIH as a contribution from the National Center for Gly-comics and Glycoproteomics at Indiana University.

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Authors and Affiliations

  1. Department of Chemistry, Indiana University, Bloomington, IN, USA

    Yehia Mechref, Milan Madera & Milos V. Novotny

Authors
  1. Yehia Mechref
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  2. Milan Madera
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  3. Milos V. Novotny
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Editor information

Editors and Affiliations

  1. Fundamental & Computational Sciences Directorate, Pacific Northwest National Laboratory, Richland, WA, USA

    Mary S. Lipton

  2. Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA, USA

    Ljiljana Paša-Tolic

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© 2009 Humana Press, a part of Springer Science+Business Media, LLC

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Mechref, Y., Madera, M., Novotny, M.V. (2009). Assigning Glycosylation Sites and Microheterogeneities in Glycoproteins by Liquid Chromatography/Tandem Mass Spectrometry. In: Lipton, M.S., Paša-Tolic, L. (eds) Mass Spectrometry of Proteins and Peptides. Methods In Molecular Biology, vol 492. Humana Press. https://doi.org/10.1007/978-1-59745-493-3_9

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  • DOI: https://doi.org/10.1007/978-1-59745-493-3_9

  • Publisher Name: Humana Press

  • Print ISBN: 978-1-934115-48-0

  • Online ISBN: 978-1-59745-493-3

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