Strategies for Differential Proteomic Analysis by Liquid Chromatography-Mass Spectrometry Public Deposited

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  • March 21, 2019
Creator
  • Stobaugh, Jordan Thomas
    • Affiliation: College of Arts and Sciences, Department of Chemistry
Abstract
  • As the field of proteomics has evolved, the analytical techniques used in this field of research has also evolved, transitioning from primarily gel-based techniques to separations based on liquid chromatography. Enzymatic digestion is often a first step for comprehensive analysis, known as bottom-up proteomics, but this comes at a cost of greatly increasing the sample complexity. Analysis of intact proteins, or top-down proteomics, is not without difficulties either as both the chromatography and mass spectrometry are more challenging. Various single and multidimensional techniques have been used to increase the peak capacity for bottom-up approaches, as, for the time being, they appear better suited for comprehensive proteomic analyses. The focus of Chapter 2 is determining what is possible given commercial instrumentation and common analytical methods for a bottom-up analysis. A single- and multidimensional separation were compared in the analysis of differentially grown yeast samples. These same samples were used in Chapter 3 in a pre-fractionation approach in which the intact protein samples were fractionated, then digested. The digested fractions were then analyzed by LC/MS to determine protein identity and relative abundance. The work presented in Chapter 5 relies on the methods developed in Chapter 3 in the differential proteomic analysis of a beta-arrestin 1,2 double-knockout. Lastly, in an effort to increase the number of protein identifications without substantially increasing analysis time, new instrumentation was designed to facilitate chromatography at pressures not attainable with commercial instrumentation. This was the focus of Chapter 4 where a nanoAcquity UPLC was modified for XUPLC operation, allowing for an increase in backpressure from 10,000 psi to 40,000 psi. With the increase in back pressure, columns with more efficiency either through smaller particle diameters or increased length could be used in the analysis of peptide samples.
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  • In Copyright
Advisor
  • Jorgenson, James
Degree
  • Doctor of Philosophy
Degree granting institution
  • University of North Carolina at Chapel Hill
Graduation year
  • 2012
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