UCLA

Proteomics has flourished as a result of major advances in computational power, data storage technologies, and network architecture, all of which enable the acquisition, handling, and processing of enormous data sets. As mass spectrometers continue to break resolution and sensitivity barriers, the limitations of qualitative and quantitative proteomic experiments will no longer result from instrumentation constraints, but rather from the quality and availability of robust sample preparation methods and post-processing algorithms. My dissertation focuses on the development of technologies in experimental proteomic workflows that precede and that follow mass spectrometric analysis - areas that are integral to the future of proteomics.

Enhanced Filter Aided Sample Preparation (eFASP) incorporates Tween-20 for plastic passivation, and n-octylglucoside and deoxycholic acid for digestion enhancement. In E. coli, eFASP reduces sample losses associated with normal FASP by almost 300%, and significantly increases hydrophobic protein representation when compared to normal FASP and to a routine precipitation-based sample preparation. eFASP is validated in the analysis of the ultra-stable proteinaceous sheath of Methanospirillum hungatei. eFASP and label-free quantitative analysis revealed a 40.7 kDa polypeptide (Q2FRN9) encoded by Mhun_2271 as the predominant sheath component by 10-fold.

eFASP is combined with sample fractionation methods and software tools for the quantitative analysis of bacterial and human samples. Hydrophilic interaction chromatography (HILIC) enables small-scale fractionation for less complex samples: Syntrophus aciditrophicus, Anaerobaculum hydrogeniformans, and melanoma-derived exosomes. S. aciditrophicus and A. hydrogeniformans are profiled for proteome shifts resulting changes in carbon source. Treatment of melanoma cells with B-Raf inhibitor, PLX-4720, results in the packaging of two tyrosine kinases, VEGFR and PDGFR-B, into secreted exosomes. Upregulation of these receptor tyrosine kinases has been found to be a result of the release of feedback inhibition following treatment with MAPK targeted therapeutics.

Complex whole-cell proteome analysis is effected by high-resolution electrostatic-repulsion hydrophilic interaction chromatography (ERLIC) fractionation of eFASP-processed samples. eFASP-ERLIC facilitated the quantitative analysis of normal and ATM-deficient human lymphoblastoid cell lines (LCLs) exposed to ionizing radiation (IR) and to YEL2, a potent radiomitigator. Exposure to IR resulted in the durable upregulation of NRF2-oxidative stress response components in Normal LCLs, while thrombin signaling and pro-survival proteins were upregulated in ATM-deficient LCLs. YEL2 treatment upregulated DNA repair components and enriched pro-survival pathways, which implicate the involvement of multiple processes in YEL2-mediated radioresistance.