INVESTIGATION OF PROTEIN PRODUCTS UNDER DIFFERENT DIGESTION CONDITIONS

Date

2024-1-25

Author

Bilgen, Dila

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The proteomics field investigates proteins qualitatively and quantitatively. The peptide-centric approach has become a widely applied technique in clinical biomarker studies based on quantitative mass spectrometry analysis. The fragmentation of proteins into peptides is a crucial part of the peptide-centric approach. Proteins are fragmented into peptides via a digestion process, which involves the steps of dissolving, denaturing, reducing, alkylating, and proteolytically cleaving the protein. Biomarker studies apply these steps to the proteins extracted from several sources, such as human plasma and serum. The literature searched for digestion procedures for human plasma and serum showed that there are numerous methods, including different reagents, reagent concentrations, incubation temperatures, and durations. However, the literature search suggested no implications for the effects of varying digestion methods on protein products. Therefore, this study systematically investigates the effects of different digestion procedures employed in the literature on protein products. Initially, digestion procedures from the scanned articles were filtered according to their frequency of use, employed reagents and their concentrations, incubation temperatures, and durations. Ten methods representing the literature search were chosen accordingly. Consequently, Alpha-2-Macroglobulin, a protein present in human blood, was digested through ten selected methods. Peptide derivatives obtained from different digestions were analyzed using a high-resolution mass spectrometer. Cleavage efficiency and artificial modification formation in peptide forms were compared to investigate the digestion efficiencies of ten selected methods. In addition, the effects of each step in the digestion procedure were compared. To accomplish this, conditions of a model protein, BSA, were investigated step by step using spectrophotometric methods. This study uncovered that different digestion procedures were not equivalent; their digestion efficiency differed, and they formed different artificial modifications on peptides. In conclusion, digestion procedures were found to be a variation source for clinical biomarker studies.

Subject Keywords

Bottom-Up Proteomics, In-Solution Protein Digestion, Alpha-2- macroglobulin, Spectroscopic Methods, Artificial Modifications

URI

https://hdl.handle.net/11511/108857

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Graduate School of Natural and Applied Sciences, Thesis