Quantitative Proteomic Analysis of Intractable Samples

Doctoral Dissertation

Abstract

Proteins regulate many cellular functions, and analyzing the presence and abundance of proteins in biological samples are central focuses in proteomics. The discovery and validation of proteins as biomarkers, pathways, and drug targets for various diseases can be accomplished using mass spectrometry-based proteomics. The mass spectrometer is a primary player in many proteomic experiments; however, the results obtained from the mass spectrometer in proteomic experiments are limited by the quality and quantity of the sample.

Regardless of the analysis approach used, a high-quality sample is critical for a successful experiment. Proteomic analyses depend on the sample containing proteins to analyze. There are several challenges in proteomic sample preparation and mass spectrometry analysis. Protein must be made readily accessible via lysis and extraction from the tissue or cells. Removal of natural contaminants and introduced detergents is necessary. Contaminants such as lipids, SDS or other detergents, and salts will interfere with enzymatic digestion, separations and mass spectrometry, sometimes damaging instruments and irreversibly ruining columns. Sample preparation approaches that are excessively time-consuming, incur massive sample losses, or fail to prevent irreversible damage require modification or replacement.

This document demonstrates my focus on sample preparation strategies for bottom-up mass spectrometry-based proteomics. The samples presented here provide unique analysis challenges. Much of the work described here focuses on samples that are easily lost, require specialized techniques, or contain high levels of natural contaminants that interfere with the proteomic workflow. Developing and applying methods for the use of such intractable samples provides opportunities for advancement of scientific discoveries in proteomics. The results of my method development efforts include the first proteomic analysis of individual tumor mimics, a novel drug evaluation technique, and a low-cost, widely available method for the analysis of protein from adipose tissue depots.

Attributes

Attribute NameValues
Author Peter E. Feist
Contributor Norman J. Dovichi, Committee Member
Contributor Sharon Stack, Committee Member
Contributor Amanda B. Hummon, Research Director
Degree Level Doctoral Dissertation
Degree Discipline Chemistry and Biochemistry
Degree Discipline Integrated Biomedical Sciences
Degree Name PhD
Defense Date
  • 2017-04-19

Submission Date 2017-06-28
Record Visibility and Access Public
Content License
  • All rights reserved

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