Towards a Greater Understanding of Post-Translational Modification of Transcription Factor, ArsR, in Helicobacter pylori via Bottom-Up Proteomics: Methods Development for Electrospray and Nanospray Approaches
Date Thesis Awarded
Honors Thesis -- Open Access
Bachelors of Science (BS)
Dr. John C. Poutsma
Dr. Mark H. Forsyth
Dr. Christopher J. Abelt, Dr. Rachel E. O'Brien
Studies presented here work towards the analysis of post-translational modification of the essential ArsR transcription factor of Helicobacter pylori. A human health hazard, H. pylori contributes to the development of gastric cancer and peptic ulcers in its hosts. It is of interest to study the mechanisms that allow the bacterium to survive long term in the harsh stomach environment. The ArsRS signaling pathway mediates the repression of sabA, the gene coding for the adhesin protein, SabA, in acidic conditions. The repression is dependent on histidine kinase, ArsS, but independent of phosphorylation of ArsR at the canonical aspartic acid 52 (D52). To begin comparing the repression mechanism in wild-type and mutant (D52E or D52N) H. pylori under neutral and acidic conditions, preliminary bottom-up proteomic investigations were conducted via LC-MS/MS.
Data-dependent mass spectrometric analyses of proteolytic (Asp-N and trypsin) digests of purified ArsR protein were performed with an ESI-LTQ linear ion trap. Ultimately, SEQUEST identified the possible phosphorylation sites, D47, D52, and D59, with high confidence from the CID product ion spectra of the purified ArsR peptides. A shotgun proteomic analysis of H. pylori (26695) was also conducted, but it was determined that the combined low-abundance of ArsR and low signal imparted by ESI preclude PTM analysis of similar shotgun samples. In addition, nanospray and electrospray methods were developed for these and future analyses. Drastic improvements in ionization efficiency and signal were observed with the nanospray ionization source in comparison to electrospray. Reliable nano-flow HPLC and nanospray ionization will be essential for future proteomic studies and the continuation of this work.
Gozzo, Theresa, "Towards a Greater Understanding of Post-Translational Modification of Transcription Factor, ArsR, in Helicobacter pylori via Bottom-Up Proteomics: Methods Development for Electrospray and Nanospray Approaches" (2017). Undergraduate Honors Theses. Paper 1137.