Characterization Of Glycosylation In Peptides And Proteins By Matrix Assisted Laser Desorption Ionization Mass Spectrometry PDF Download

This dissertation, "Mass Spectrometric Analysis of Selected Glycoproteins" by Chun-yu, Chan, was obtained from The University of Hong Kong (Pokfulam, Hong Kong) and is being sold pursuant to Creative Commons: Attribution 3.0 Hong Kong License. The content of this dissertation has not been altered in any way. We have altered the formatting in order to facilitate the ease of printing and reading of the dissertation. All rights not granted by the above license are retained by the author. Abstract: Abstract of thesis entitled MASS SPECTROMETRIC ANALYSIS OF SELECTED GLYCOPROTEINS Submitted by Chan Chun Yu for the degree of Master of Philosophy at The University of Hong Kong in February 2005 Fetuin from fetal calf serum (Sigma, F6131), a well-characterized glycoprotein that has a molecular weight of 48.4 kDa and 3 N-glycosylation sites, was used as a prototypical protein to develop mass spectrometric approaches toward the identification and sequence elucidation of the N-glycosylation sites of proteins. Fetuin was digested using trypsin (porcine) and deglycosylated using peptide N-glycosidase F (PNGase-F) prior to matrix-assisted laser desorption/ionization-time-of-flight (MALDI-TOF) mass spectrometric analysis. For peptide mass fingerprinting, the experimental peak list obtained from the MALDI-TOF mass spectrum of the sample was compared with the theoretical peak list obtained from the National Center for Biotechnology Information sequence database. The identification of each N-glycosylation site was based on the 0.98-Da mass increase observed upon hydrolysis by the amidase PNGase-F of the glycosylamine linkage of each N-glycosylated asparagine, which formed an aspartic acid residue. The identities of the glycopeptides were verified through isotopic pattern analysis using the proposed amino acid composition and the ISOPRO shareware program. All the 3 potential N-glycosylation sites of fetuin were identified successfully in accordance with published results. The peptide sequence coverage was 75%. The developed strategy was then applied to map the N-glycosylation sites of the severe acute respiratory syndrome spike S glycoprotein, a novel glycoprotein that requires a more complex analysis for its total 23 potential N-glycosylation sites. The proposed peptide sequence and its potential N-glycosylation sites were elucidated using tandem mass spectrometry under low-energy collision-induced dissociation. The partial peptide sequence was determined by computing the differences in the mass-to-charge ratios between consecutive b- or y- fragment ions. The sequence tags generated this way were used to confirm the exact sites of N-glycosylation. The effects that the sequential specific enzymatic digestion process, the MALDI matrix, the sample clean-up process, and the enrichment methods have on protein sequence coverage were also examined. For the spike S glycoprotein, a sequence coverage of 52% was obtained. Within the covered peptide sequences, 17 of the 23 potential sites were detected (except for N7, 8, 13, 15, 16, and 20); 6 of them were novel N-glycosylation sites. DOI: 10.5353/th_b3147942 Subjects: Glycoproteins - Analysis Spectrum analysis