Post-translational modifications (PTMs) of proteins can have significant effects on different biological processes in which the proteins are involved. Analysis of protein post-translational modifications (PTMs) provides crucial information that elucidates molecular mechanisms of diseases and leads to new diagnosis paradigms and treatment strategies. This dissertation serves as a record of our efforts to identify and quantify two types of protein PTMs by mass spectrometry: carbonylation and glycosylation. Carbonylation of proteins induced by acrolein is a non-enzymatic chemical modification. Through Michael addition, acrolein adds a carbonyl group to the side chain of cysteine, lysine, or arginine. Acrolein can be generated in the liver as a metabolite of chemicals such as ally alcohol and cyclophosphamide; and as a result, may be responsible for each molecule’s demonstrated liver toxicity. To determine the identity of the proteins selectively adducted by acrolein, rat liver microsomes were treated with acrolein under non-denature condition, then an analysis strategy combining biotin tagging of carbonylated proteins, avidin enrichment of biotinylated peptides and LC-MS/MS characterization was utilized. Proteins potentially involved in liver toxicity were characterized including enzymes involved in metabolism of acrolein. This study validates the peptide-centric approach and should facilitate an understanding of the role that protein carbonylation plays in acrolein’s toxicity within the liver. The glycosylation profile of prostate specific antigen (PSA) is altered in patients suffering from prostate cancer. Characterizing the glycoforms of PSA in serum that are specific to the disease state could potentially be a diagnostic method of prostate cancer with high specificity. As proof of principle, two different commercially-available PSA samples with different glycosylation profiles were analyzed. A bottom-up approach was applied for glycol-quantification of a protein, which included protein digestion, HILIC SPE purification of glycopeptides, and detection of glycopeptides via reversed-phase (RP) LC-MS/MS. By revealing relative abundances of twenty-eight differential glyco-isoforms, this pilot study confirms the potential utility of a peptide-centered approach for detecting and quantifying different glycoforms of a specific protein.
Zhu, Yiying,
"Mass Spectrometric Analysis of Protein Carbonylation and Glycosylation"
(2014).
Chemistry Theses and Dissertations.
Brown Digital Repository. Brown University Library.
https://doi.org/10.7301/Z0Z899RH
