SENSITIVE GLOBAL PROFILING OF PROTEOLYSIS IN COMPLEX BIOCHEMICAL MIXTURES

这个子项目是众多研究子项目之一

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Proteolysis plays an important role in the regulation of diverse biological processes. Unfortunately, current methods for monitoring proteolytic events in complex samples suffer from serious limitations and lag far behind those typically used for proteomic profiling of other post-translational modifications such as phosphorylation and ubiquitination. The aim of our work is development and application of a novel method for global profiling of proteolysis in complex biochemical mixtures that is sensitive, robust, and general. This method is based on the use of an engineered peptide ligase to selectively label protein N-termini in cell lysates, serum, and other complex biochemical mixtures. The label permits 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 is indicative of a proteolytic event at the sequenced amino acid site. This method is being applied to the study of proteolysis in several different biological systems including cellular programmed cell death, or apoptosis, inflammation, and human serum. This work will shed new light on the biology of apoptosis, inflammation, serum, and the relationship between proteolysis in each of these systems in health and disease. Furthermore, this work will enable continued refinement of a novel proteomic method that we hope 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.