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 at least 280 proteins are known to undergo proteolytic processing as part of the biochemical events leading to programmed cell death. Adding to this complexity, the nature of the apoptotic response can vary depending on the apoptotic stimulus and the cell type in a manner that cannot always be predicted. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations.
The aim of this work is to establish a novel method for global profiling of proteolysis in complex biochemical mixtures that is sensitive, robust, and general. This method will be based on the use of an engineered peptide ligase to selectively label protein N-termini in cell lysates. The label will permit affinity purification and enrichment of N-terminal peptides for subsequent sequencing by tandem mass spectrometry. Identification of N-termini present in experimental samples and absent in control samples will be indicative of a proteolytic event at the sequenced amino acid site. Analysis of proteolysis in apoptosis will at first be used as a model system for method development. A matured version of the method will then be applied to survey the diversity of proteolysis patterns in apoptosis elicited by different stimuli and in different cell types, and to identify new targets of proteolysis in apoptosis. This work will shed new light on the biology of apoptosis and will establish a novel proteomic method that will find application to the study of proteolysis in other areas of biology. The UCSF Mass Spectrometry Facility will provide the mass spectrometry instrumentation, software, and expertise essential for the success of this work.
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