Chemistry & Biochemistry
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Item INVESTIGATION BY MASS SPECTROMETRY OF THE UBIQUITOME AND PROTEIN CARGO OF EXOSOMES DERIVED FROM MYELOID-DERIVED SUPPRESSOR CELLS(2016) Adams, Katherine R.; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Exosomes released by myeloid-derived suppressor cells (MDSC) are 30 nm in diameter extracellular vesicles that have been shown to carry biologically active proteins as well as ubiquitin molecules. Ubiquitin is known to have many functions, including involvement in the formation of exosomes, although the exact role is highly contested. In the study reported here, the proteome and ubiquitome of MDSC exosomes has been investigated by bottom-up proteomics techniques. This report identifies more than 1000 proteins contained in the MDSC exosome cargo and 489 sites of ubiquitination in more than 300 ubiquitinated proteins based on recognition of glycinylglycine tagged peptides without antibody enrichment. This has allowed extensive chemical and biological characterization of the ubiquitinated cohort compared to that of the entire protein cargo to support hypotheses on the role of ubiquitin in exosomes.Item Analysis of Intact Proteins in Complex Mixtures(2013) Dhabaria, Avantika; Fenselau, Catherine; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our goal is to develop an effective work flow for analysis of intact proteins in a complex mixture using the LC-LTQ-Orbitrap XL. Intact protein analysis makes the entire sequence available for characterization, which allows for the identification of isoforms and post translational modifications. We focus on developing a method for top-down proteomics using a high-resolution, high mass accuracy analyzer coupled with bioinformatics tools. The complex mixtures are fractionated using 1-dimensional reversed-phase chromatography and basic reversed- phase, and open tubular electrophoresis. The analysis of intact proteins requires various fragmentation methods such as collisional induced dissociation, high energy collisional dissociation, and electron transfer dissociation. This overall method enables us to analyze intact proteins, providing a better understanding of protein expression levels and post transitional modification information. We have used standard proteins to optimize HPLC conditions and to compare three methods for ion activation and dissociation. Furthermore, we have extended the method to analyze low mass proteins in MCF7 cytosol and in E. coli lysate as a model complex mixture. We have applied this strategy to identify and characterize proteins from extracellular vesicles (EVs) shed by murine myeloid-derived suppressor cells (MDSC). MDSCs suppress both innate and adaptive immune responses to tumor growth and prevent effective immunotherapy. Recently some of the intercellular immunomodulatory effects of MDSC have been shown to be propagated by EVs. Top-down analysis of intact proteins from these EVs was undertaken to identify low mass protein cargo, and to characterize post-translational modifications.Item Microwave-Supported Acid Hydrolysis for Proteomics(2012) Cannon, Joe; Fenselau, Catherine; Biochemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Our goal is to develop, optimize and demonstrate workflows that incorporate rapid Asp-selective chemical proteolysis into proteomic studies of complex mixtures. This can be further divided into several specific aims. The first aim is to develop and optimize the sample preparation, mass spectrometric, and bioinformatic methods required for complex mixture analysis of peptides resulting from acid digestion both in solution and in polyacrylamide gels. Second, the optimized methods will be applied to three model systems. In the first application, the large peptides derived from microwave-supported acid hydrolysis of human ribosomes isolated from MCF-7 breast cancer cells are analyzed. Secondly, acid hydrolysis will be applied to characterize Lys63 linkages in polyubiquitins. Finally, all the above methods will be combined for the analysis of extracellular vesicles shed by myeloid derived suppressor cells from a murine mammary carcinoma model. After optimizing the mass spectrometric and bioinformatic methods required for analysis of peptides resulting from acid hydrolysis, the most comprehensive analysis using this digestion technique to date was achieved both for in gel and in solution analysis. In gel digestion resulted in identification of over twelve hundred peptides representing 642 proteins, and in solution digestion via mass biased partitioning allowed identification of over 300 proteins. Mass biased partitioning also resulted in two distinct peptide populations from the high and low mass analyses implemented. Nearly 90% of the predicted human ribosomal proteins were identified after acid hydrolysis. High resolution analysis of both precursor and product ions resulted in an average sequence coverage of 46% among identified proteins. It was also demonstrated that microwave-supported acid hydrolysis facilitates a more informative method for analysis of Lys63 linked polyubiquitin. After acid hydrolysis, ~629 Da mass shifts were found to be indicative of isopeptides. These isopeptides were easily identified from complex mixtures using tandem mass spectrometry and diagnostic b ions. Extracellular vesicles from a murine carcinoma model were then analyzed using in gel microwave-supported acid hydrolysis, mass biased partitioning after in solution digestion, and the sample was interrogated for the presence of ubiquitinated peptides.Item STRUCTURAL STUDIES OF THE PEANUT ALLERGEN PROTEIN ARA H 2(2010) Li, Jinxi; FENSELAU, CATHERINE; Chemistry; Digital Repository at the University of Maryland; University of Maryland (College Park, Md.)Presented in this dissertation are comprehensive studies of the structures of the peanut allergen protein Ara h 2 and the effect of food processing (roasting) on it. A detailed elucidation of the primary structure and PTM of Ara h 2 from the raw peanuts has been described. Ara h 2 isoforms were purified and cleaved via microwave accelerated trypsin digestion. The peptide mixtures were analyzed by LC-MS/MS and targeted CID. De novo sequencing of the MS/MS spectra revealed the protein sequence of each Ara h 2 isoform. Several hydroxyproline sites have been discovered while disulfide bond structures have been partially determined. Using anti-Ara h 2 antibodies, Western blotting of 1-D gels of the raw and dark roasted peanuts was carried out in order to characterize the changes of Ara h 2 between these two samples. The result indicates that Ara h 2 may present in a much heavier form in the roasted peanuts, possibly due to crosslinking and aggregation with other proteins. Subsequent LC-MS/MS studies of trypsin digestion of five gel pieces (>100, 100-50, 50-25, 25-16 kDa) from 1-D gels of the raw and dark roasted peanuts suggests that roasting process causes the crosslinking of Ara h 2 with other proteins. This supports our results from the immunological studies.