Site determination of protein glycosylation based on digestion with immobilized nonspecific proteases and Fourier transform ion cyclotron resonance mass spectrometry.
An improved method for site-specific characterization of protein glycosylation has been devised using nonspecific digestion with immobilized pronase combined with Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS). This procedure was demonstrated using ribonuclease B (RNase B) and kappa-casein (kappa-csn) as representative N-linked and O-linked glycoproteins, respectively. Immobilization of the pronase enzymes facilitated their removal from the glycopeptide preparations, and was found to prevent enzyme autolysis while leaving the proteolytic activities of pronase intact. Increased digestion efficiency, simplified sample preparation, and reduced sample complexity were consequently realized. To supplement this technique, a refined glycopeptide search algorithm was developed to aid in the accurate mass based assignment of N-linked and O-linked glycopeptides derived from nonspecific proteolysis. Monitoring the progress of glycoprotein digestion over time allowed detailed tracking of successive amino acid cleavages about the sites of glycan attachment, and provided a more complete protein glycosylation profile than any single representative time point. This information was further complemented by tandem MS experiments with infrared multiphoton dissociation (IRMPD), allowing confirmation of glycopeptide composition. Overall, the combination of immobilized pronase digestion, time course sampling, FTICR-MS, and IRMPD was shown to furnish an efficient and robust approach for the rapid and sensitive profiling of protein glycosylation.
Clowers
Brian H
BH
Department of Chemistry, University of California, Davis, Davis, California 95616, USA.
Dodds
Eric D
ED
Seipert
Richard R
RR
Lebrilla
Carlito B
CB
D000465:Algorithms; D000818:Animals; D002364:Caseins / Q000737:chemistry; D002417:Cattle; D004800:Enzymes, Immobilized; D005583:Fourier Analysis; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D013058:Mass Spectrometry / Q000379:methods; D011402:Pronase / Q000737:chemistry*; D012260:Ribonucleases / Q000737:chemistry