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Open Access Publications from the University of California

Scholarly Works (5 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2012)

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.

    Cover page: High throughput analysis of proteome perturbations induced by radiation, radiomitigators and chemotherapeutics
    • Article
    • Peer Reviewed
    UCLA Previously Published Works (2014)
    The integrity of quantitative proteomic experiments depends on the reliability and the robustness of the protein extraction, solubilization, and digestion methods utilized. Combinations of detergents, chaotropes, and mechanical disruption can yield successful protein preparations; however, the methods subsequently required to eliminate these added contaminants, in addition to the salts, nucleic acids, and lipids already in the sample, can result in significant sample losses and incomplete contaminant removal. A recently introduced method for proteomic sample preparation, filter-aided sample preparation (FASP), cleverly circumvents many of the challenges associated with traditional protein purification methods but is associated with significant sample loss. Presented here is an enhanced FASP (eFASP) approach that incorporates alternative reagents to those of traditional FASP, improving sensitivity, recovery, and proteomic coverage for processed samples. The substitution of 0.2% deoxycholic acid for urea during eFASP digestion increases tryptic digestion efficiency for both cytosolic and membrane proteins yet obviates needed cleanup steps associated with use of the deoxycholate sodium salt. For classic FASP, prepassivating Microcon filter surfaces with 5% TWEEN-20 reduces peptide loss by 300%. An express eFASP method uses tris(2-carboxyethyl)phosphine and 4-vinylpyridine to alkylate proteins prior to deposition on the Microcon filter, increasing alkylation specificity and speeding processing.
      Cover page: Enhanced FASP (eFASP) to Increase Proteome Coverage and Sample Recovery for Quantitative Proteomic Experiments
      • Article
      • Peer Reviewed
      UCLA Previously Published Works (2017)
      Enhanced Filter Aided Sample Preparation (eFASP) incorporates plastics passivation and digestion-enhancing surfactants into the traditional FASP workflow to reduce sample loss and increase hydrophobic protein representation in qualitative and quantitative proteomics experiments. Resulting protein digests are free of contaminants and can be analyzed directly by LC-MS.
        Cover page: Improving Proteome Coverage and Sample Recovery with Enhanced FASP (eFASP) for Quantitative Proteomic Experiments
        • Article
        • Peer Reviewed
        UCLA Previously Published Works (2019)
        Syntrophy is essential for the efficient conversion of organic carbon to methane in natural and constructed environments, but little is known about the enzymes involved in syntrophic carbon and electron flow. Syntrophus aciditrophicus strain SB syntrophically degrades benzoate and cyclohexane-1-carboxylate and catalyses the novel synthesis of benzoate and cyclohexane-1-carboxylate from crotonate. We used proteomic, biochemical and metabolomic approaches to determine what enzymes are used for fatty, aromatic and alicyclic acid degradation versus for benzoate and cyclohexane-1-carboxylate synthesis. Enzymes involved in the metabolism of cyclohex-1,5-diene carboxyl-CoA to acetyl-CoA were in high abundance in S. aciditrophicus cells grown in pure culture on crotonate and in coculture with Methanospirillum hungatei on crotonate, benzoate or cyclohexane-1-carboxylate. Incorporation of 13 C-atoms from 1-[13 C]-acetate into crotonate, benzoate and cyclohexane-1-carboxylate during growth on these different substrates showed that the pathways are reversible. A protein conduit for syntrophic reverse electron transfer from acyl-CoA intermediates to formate was detected. Ligases and membrane-bound pyrophosphatases make pyrophosphate needed for the synthesis of ATP by an acetyl-CoA synthetase. Syntrophus aciditrophicus, thus, uses a core set of enzymes that operates close to thermodynamic equilibrium to conserve energy in a novel and highly efficient manner.
          Cover page: Syntrophus aciditrophicus uses the same enzymes in a reversible manner to degrade and synthesize aromatic and alicyclic acids
          • Article
          • Peer Reviewed
          UCLA Previously Published Works (2016)
          Syntrophus aciditrophicus is a model syntrophic bacterium that degrades key intermediates in anaerobic decomposition, such as benzoate, cyclohexane-1-carboxylate, and certain fatty acids, to acetate when grown with hydrogen-/formate-consuming microorganisms. ATP formation coupled to acetate production is the main source for energy conservation by S. aciditrophicus However, the absence of homologs for phosphate acetyltransferase and acetate kinase in the genome of S. aciditrophicus leaves it unclear as to how ATP is formed, as most fermentative bacteria rely on these two enzymes to synthesize ATP from acetyl coenzyme A (CoA) and phosphate. Here, we combine transcriptomic, proteomic, metabolite, and enzymatic approaches to show that S. aciditrophicus uses AMP-forming, acetyl-CoA synthetase (Acs1) for ATP synthesis from acetyl-CoA. acs1 mRNA and Acs1 were abundant in transcriptomes and proteomes, respectively, of S. aciditrophicus grown in pure culture and coculture. Cell extracts of S. aciditrophicus had low or undetectable acetate kinase and phosphate acetyltransferase activities but had high acetyl-CoA synthetase activity under all growth conditions tested. Both Acs1 purified from S. aciditrophicus and recombinantly produced Acs1 catalyzed ATP and acetate formation from acetyl-CoA, AMP, and pyrophosphate. High pyrophosphate levels and a high AMP-to-ATP ratio (5.9 ± 1.4) in S. aciditrophicus cells support the operation of Acs1 in the acetate-forming direction. Thus, S. aciditrophicus has a unique approach to conserve energy involving pyrophosphate, AMP, acetyl-CoA, and an AMP-forming, acetyl-CoA synthetase. Bacteria use two enzymes, phosphate acetyltransferase and acetate kinase, to make ATP from acetyl-CoA, while acetate-forming archaea use a single enzyme, an ADP-forming, acetyl-CoA synthetase, to synthesize ATP and acetate from acetyl-CoA. Syntrophus aciditrophicus apparently relies on a different approach to conserve energy during acetyl-CoA metabolism, as its genome does not have homologs to the genes for phosphate acetyltransferase and acetate kinase. Here, we show that S. aciditrophicus uses an alternative approach, an AMP-forming, acetyl-CoA synthetase, to make ATP from acetyl-CoA. AMP-forming, acetyl-CoA synthetases were previously thought to function only in the activation of acetate to acetyl-CoA.
            Cover page: Pyrophosphate-Dependent ATP Formation from Acetyl Coenzyme A in Syntrophus aciditrophicus, a New Twist on ATP Formation
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