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Identifying the Glycosylation Sites and Site‐specific Carbohydrate Heterogeneity of Glycoproteins by Matrix‐assisted Laser Desorption/Ionization Mass Spectrometry
Author(s) -
Yang Yi,
Orlando Ron
Publication year - 1996
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/(sici)1097-0231(19960610)10:8<932::aid-rcm595>3.0.co;2-x
Subject(s) - chemistry , glycosylation , mass spectrometry , glycoprotein , matrix assisted laser desorption/ionization , peptide , chromatography , glycopeptide , oligosaccharide , protein mass spectrometry , biochemistry , carbohydrate , desorption , tandem mass spectrometry , organic chemistry , adsorption , antibiotics
Abstract A sensitive and facile method is described to identify the glycosylation sites and site‐specific heterogeneity in the carbohydrate attached to glycoproteins. In this procedure, the peptide backbone of the glycoprotein is cleaved enzymatically. The resulting peptide/glycopeptide mixture is divided into three fractions. The first is analyzed directly by matrix‐assisted laser desorption/ionization mass spectrometry (MALDI‐MS), while the other two aliquots are analyzed by MALDI‐MS after enzymatic release of the N ‐linked chains and the N ‐ and O ‐linked chains. Comparison of these MALDI mass spectra provides the molecular weight of each carbohydrate side chain and of the peptide to which it was attached. This information combined with the amino acid sequence of the protein identifies the glycosylation sites, and provides information concerning site‐specific oligosaccharide heterogeneity. This approach does not require time‐consuming liquid chromatographic separations and can be performed on as little as 10 pmol of glycoprotein. Thus, our approach is faster and simpler than procedures currently used for glycosylation site mapping, and may offer a slight sensitivity advantage.