Assigning glycosylation sites and microheterogeneities in glycoproteins by liquid chromatography/tandem mass spectrometry

• Published in: Methods in molecular biology (Clifton, N.J.) Vol. 492; p. 161
• Main Authors: Mechref, Yehia, Madera, Milan, Novotny, Milos V
• Format: Journal Article
• Language: English
• Published: United States 2009
• Subjects: alpha-Fetoproteins - analysis; alpha-Fetoproteins - chemistry; alpha-Fetoproteins - metabolism; Amino Acid Sequence; Animals; Binding Sites; Cattle; Chromatography, Affinity; Chromatography, Liquid - methods; Glycopeptides - analysis; Glycopeptides - chemistry; Glycopeptides - metabolism; Glycoproteins - analysis; Glycoproteins - chemistry; Glycoproteins - metabolism; Glycosylation; Lectins - chemistry; Molecular Sequence Data; Silicon Dioxide - chemistry; Tandem Mass Spectrometry - methods; Trypsin - metabolism
• DOI: 10.1007/978-1-59745-493-3_9
• ISSN: 1064-3745

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 microheterogeneities 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.