Multidimensional separation of intact proteins for differential proteomics employing top-down and bottom-up proteomic strategies Public Deposited

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  • March 21, 2019
Creator
  • Richardson, Brenna McJury
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • Differential proteomics, sometimes referred to as differential expression proteomics, is a subset of proteomics that attempts to identify and quantify changes in protein expression between multiple samples. One of the major challenges associated with any proteomics experiment is sample complexity. The traditional method of analysis involves a gel-based separation of intact protein for a quantitative comparison followed by analysis by mass spectrometry to identify differentially expressed proteins. While this method does have the resolving power to accommodate the sample complexity encountered in proteomics, there has been a shift towards liquid chromatography-based separations to improve automation, reduce sample bias, and more easily couple the separation to mass spectrometry. In Chapter 2, an on-line liquid chromatographic separation strategy was developed similar in nature to the traditional gel-based approach and was applied to the analysis of differential yeast samples. It involved the on-line 2D separation of intact protein followed by MS analysis and fraction collection. A digestion of select fractions was performed to identify differentially expressed protein in a bottom-up manner. The experiment is top-down as it uses the intact protein MS signal for quantification and determination of the intact protein molecular weight, but is bottom-up in that proteins are identified following enzymatic digestion. This methodology is applied in Chapter 6 to study the differential expression of proteins in both wild-type and beta-arrestin 1,2 double knockout mouse embryonic fibroblast cells. Chapters 3, 4 and 5 focus on examining the correlation between experiments performed in either a top-down or bottom-up manner. A digestion of the complete sample set was performed in Chapter 3 in a conventional, bottom-up only experiment. The analysis discussed in Chapter 4 directly compares the differential expression of protein determined by a top-down experiment to the expression from a bottom-up experiment. To facilitate this comparison, intact proteins were initially fractionated and then split such that each fraction was analyzed by both proteomic methods. In Chapter 5, intact proteins were fractionated prior to digestions in an attempt to improve both identification and quantification of proteins. The differential expressions from all forms of analysis of the same sample set were analyzed and compared.
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  • "... in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Department of Chemistry."
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  • Jorgenson, James
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  • Chapel Hill, NC
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  • Open access
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