Date of Award

8-2020

Document Type

Campus Access Thesis

Degree Name

Master of Science (MS)

Department

Chemistry

First Advisor

Jason Evans

Second Advisor

Marianna Torok

Third Advisor

Daniel Dowling

Abstract

Liquid chromatography electrospray tandem mass spectrometry has become the method of choice for quantitative proteomics studies seeking to measure differential protein expression patterns. The data-dependent workflows for large discovery-based studies have been successful in identifying potential protein targets worthy of further investigation. Due to reproducibility issues that plague these studies, the current recommendations among practitioners are to perform targeted proteomics experiments to validate the results of large scale discovery-based experiment. In this work, a targeted proteomics workflow for the quantification of GADD45α, CDKN1A, and p53 proteins that are known to be overexpressed in response to a DNA repair event, was constructed based on the TOMAHAQ method developed by Gygi. The accuracy and precision of the quantification of these proteins in the presence of a complex interfering proteome were evaluated and compared directly to similar data obtained from a typical data-dependent SPS10 MS3 approach utilizing the “SPS Mass Matches [%] Threshold” filter in Protein Discoverer. Using the TOMAHAQ method, the data for most abundant protein, GADD45α, showed excellent accuracy and precision down to 16 fmol in the TMT6-131 channel injected on the column, even in the presence of 1.5 μg of an interfering HeLa proteome in this channel. Inaccuracies due to co-isolated HeLa peptides plagued the data for the tryptic peptides of the other two proteins. The data obtained using the data-dependent SPS10 MS3 analysis for GADD45α was also of high quality and was not significantly different than the data obtained using the TOMAHAQ method for any of the three channels that contained the interfering HeLa proteome. Our data suggest that the targeted method based on TOMAHAQ does not provide data of higher quality than the data-dependent SPS10 MS3 approach when “SPS Mass Matches [%] Threshold” filter is set to the default value of 65 %.

Comments

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