This subproject is one of many research subprojects utilizing theresources provided by a Center grant funded by NIH/NCRR. The subproject andinvestigator (PI) may have received primary funding from another NIH source,and thus could be represented in other CRISP entries. The institution listed isfor the Center, which is not necessarily the institution for the investigator.Proteolysis plays an important role in the regulation of diverse biological processes. One such process is apoptosis, in which approximately 300 proteins are known to undergo proteolytic processing as part of the biochemical events leading to programmed cell death. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations. We are developing a novel method for global profiling of proteolysis in complex biochemical mixtures that is based on the use of an engineered peptide ligase to selectively label the alpha amines protein N-termini. The label permits affinity purification and enrichment of N-terminal peptides for subsequent sequencing by tandem mass spectrometry. This approach not only enables monitoring of newly formed N-termini as a result of proteolysis is cells or serum, but also decreases sample complexity for greater proteomic coverage. We are applying this method to profile the proteolysis that occurs during apoptosis in cells, and to profile changes in serum protein levels following apoptosis. Our goal is to survey the diversity of proteolysis patterns in apoptosis elicited by different stimuli and in different cell types, to identify new targets of proteolysis in apoptosis, and to explore how levels of serum proteins change in different stages of cancer and chemotherapy. This work will shed new light on the biology of apoptosis and how apoptosis can be best exploited in chemotherapy. The work will also establish a novel proteomic method that will find application in other areas of biology.
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