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.