Glycoprotein identification and localization of O-glycosylation sites by mass spectrometric analysis of deglycosylated/alkylaminylated peptide fragments.
In-gel digestion of densely O-glycosylated proteins, an essential step in proteome analysis, is often hampered by steric hindrance of the proteases. To overcome this technical problem a simple and convenient method has been developed, which combines several advantages: (1) Approximately 70% of the oligosaccharides are cleaved without significant protein hydrolysis at the optimal reaction conditions of 70% ethylamine, and quantitative cleavage is achieved with 40% methylamine, at 50 degrees C. (2) To the unsaturated derivatives of Ser and Thr the alkylamine is added as a label of previous O-glycosylation sites. (3) The alkylaminylated protein is effectively cleaved by proteolysis. (4) The modified peptides are identified by MALDI mass spectrometry under consideration of incremental mass increases. (5) The alkylamine label is stable under MALDI post-source-decay analysis as well as in collision-induced dissociation experiments allowing sequencing and peptide localization of O-glycosylation sites. Applicability of the method is evaluated with a series of synthetic glycopeptides, the densely O-glycosylated human glycophorin A, and with the mucin MUC1 from human milk fat globule membranes.
Hanisch
F G
FG
Institute of Biochemistry, Medical Faculty of the University, Joseph-Stelzmann-Strasse 52, Köln, 50931, Germany. franz.hanisch@uni-koeln.de
Jovanovic
M
M
Peter-Katalinic
J
J
D000478:Alkylation; D000588:Amines / Q000737:chemistry; D000595:Amino Acid Sequence; D006017:Glycolipids / Q000378:metabolism; D006020:Glycopeptides / Q000032:analysis* / Q000378:metabolism; D006021:Glycophorins / Q000032:analysis; D006023:Glycoproteins / Q000378:metabolism; D006031:Glycosylation; D006801:Humans; D066292:Lipid Droplets; D009077:Mucins / Q000378:metabolism; D010446:Peptide Fragments / Q000737:chemistry* / Q000378:metabolism; D020543:Proteome / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000588:Amines; D006017:Glycolipids; D006020:Glycopeptides; D006021:Glycophorins; D006023:Glycoproteins; D009077:Mucins; D010446:Peptide Fragments; D020543:Proteome; C110367:milk fat globule; D014357:Trypsin