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University of Texas – Austin

1. -5604-9810. Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications.

Degree: Chemistry, 2018, University of Texas – Austin

Post-translational modifications (PTMs) are important for regulating protein structure and function. Despite significant progress for PTM analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS), opportunities for new method development remain. The research presented in this dissertation promotes 193 nm ultraviolet photodissociation (UVPD) as an alternative activation technique for PTM analysis with specific utility for phosphorylated and sulfated peptides. A novel de novo sequencing method with applications for unbiased PTM discovery was developed utilizing Lys-N proteolysis, N-terminal imidazolinylation, and UVPD to direct fragmentation for the formation of N-terminal ions. The N-terminal a, b, and c ions generated by UVPD were differentiated from one another by characteristic mass shifts. Sets of triplet peaks were used to distinguish N-terminal ions from confounding C-terminal ions and improve the accuracy of de novo sequencing. UVPD was evaluated for the analysis of phosphopeptide cations and anions. Negative mode analysis was advantageous for the detection of casein peptides in high phosphorylation states, while positive mode proved more robust for global phosphoproteomic analysis of HeLa and HCC70 cell lysates. Compared to collisional activation, the depth of coverage was lower using UVPD yet more extensive fragmentation and improved phosphate retention on products ions was achieved. Phosphorylation mapping by LC-UVPD-MS was carried out in the C-terminal domain (CTD) of RNA polymerase II as a function of kinase treatment, ERK2 or TFIIH, and organism, yeast or fruit fly. Single phosphorylations on Ser2 or Ser5 in the consensus heptad, YSPTSPS, were observed across all experimental conditions. Analysis of the non-consensus fruit fly CTD revealed the significance of Tyr1 and Pro residues in the +1 position relative to Ser for phosphorylation to occur. For sulfated peptides, negative mode UVPD yielded a and x ions that largely retained the labile sulfate modification, facilitating peptide sequencing and PTM localization. With appropriate MS/MS tools established, the next step towards global sulfoproteomics was the development of enrichment methods. Weak anion exchange (WAX) was applied for this purpose. Following carbamylation to neutralize primary amines which otherwise repel the anion exchanger; improved WAX retention was observed for sulfopeptides relative to a complex mixture of unmodified bovine serum albumin peptides. Advisors/Committee Members: Brodbelt, Jennifer S. (advisor), Crooks, Richard M (committee member), Dalby, Kevin N (committee member), Webb, Lauren J (committee member), Zhang, Yan J (committee member).

Subjects/Keywords: Mass spectrometry; Ultraviolet photodissociation; Proteomics; Phosphorylation; Sulfation

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APA (6th Edition):

-5604-9810. (2018). Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications. (Thesis). University of Texas – Austin. Retrieved from http://hdl.handle.net/2152/68369

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation

Chicago Manual of Style (16th Edition):

-5604-9810. “Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications.” 2018. Thesis, University of Texas – Austin. Accessed October 16, 2018. http://hdl.handle.net/2152/68369.

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation

MLA Handbook (7th Edition):

-5604-9810. “Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications.” 2018. Web. 16 Oct 2018.

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete

Vancouver:

-5604-9810. Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications. [Internet] [Thesis]. University of Texas – Austin; 2018. [cited 2018 Oct 16]. Available from: http://hdl.handle.net/2152/68369.

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation

Council of Science Editors:

-5604-9810. Advancement of photodissociation mass spectrometry methods for the analysis of protein post-translational modifications. [Thesis]. University of Texas – Austin; 2018. Available from: http://hdl.handle.net/2152/68369

Note: this citation may be lacking information needed for this citation format:
Author name may be incomplete
Not specified: Masters Thesis or Doctoral Dissertation

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