Proteomics of glycoproteins
Abstract
The objective of the work presented in this thesis was to test the concept that tryptic peptides may be used as analytical surrogates of the protein from which they were derived. Proteins in complex mixtures were digested with trypsin and classes of peptide fragments selected by affinity chromatography and lectin columns were employed to select glycopeptides. For detecting known glycoproteins in a mixture, affinity-selected glycopetpide mixtures were directly transferred to a high resolution reversed-phase chromatography column and further resolved into fractions that were collected and subjected to MALDI-TOF mass spectrometry. The presence of human transferrin in human serum was detected by matching the glycopeptide mass fingerprints from standard human transferrin to the mass fingerprint of the glycopeptide in the human serum. For identifying unknown N-glycoproteins, affinity-selected peptides were deglycosylated in order to measure the molecular weight of these peptides. Both enzymatic deglycosylation and mild chemical deglycosylation methods were tested. Carboxypeptidase was used to obtain partial sequences of the peptides. In addition, specific structure—NXS(T)—was used with molecular weights and partial sequences to identify proteins from a database employing a computer search procedure. O-GlcNac modified glycopeptides in nuclear extract were also examined.
Degree
Ph.D.
Advisors
Regnier, Purdue University.
Subject Area
Analytical chemistry
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