18O-labeling of N-glycosylation sites to improve the identification of gel-separated glycoproteins using peptide mass mapping and database searching.
Peptide mass mapping using matrix-assisted laser desorption/ionization (MALDI) mass spectrometry in conjunction with interrogation of sequence databases is a powerful tool for the identification of proteins. Glycosylated proteins often yield poor MALDI peptide maps due to shielding of proteolytic cleavage sites and the presence of modified peptides. Here we demonstrate that enzymatic removal of N-linked glycans with simultaneous partial (50%) 18O-labeling of glycosylated asparagine residues prior to proteolysis and MALDI peptide mass mapping can overcome these problems. As a result, more peptides are observed in MALDI spectra which, in turn, increases the specificity of subsequent database searches. Furthermore, the detection of a labeled peptide directly translates into partial sequence information as N-linked carbohydrates are exclusively attached to asparagine residues that form part of the NXS/T sequence. The mass of the formerly glycosylated peptide together with the NXS/T sequence pattern represents a discriminating criterion for database searching which, on average, increases the search specificity by a factor of 100. This procedure allows the unambiguous identification of glycoproteins that would otherwise require sequencing and, at the same time, enables the identification of N-glycosylation sites with higher sensitivity than previously possible.
Küster
B
B
Protein Interaction Laboratory, University of Southern Denmark, Odense M.
Mann
M
M
D000595:Amino Acid Sequence; D016208:Databases, Factual; D006023:Glycoproteins / Q000032:analysis* / Q000302:isolation & purification; D006031:Glycosylation; D007202:Indicators and Reagents; D008969:Molecular Sequence Data; D010104:Oxygen Radioisotopes; D010449:Peptide Mapping / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
D006023:Glycoproteins; D007202:Indicators and Reagents; D010104:Oxygen Radioisotopes