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. Proteolytic processing is a key post-translational modification in blood plasma and serum. Most secreted proteins are processed by proteases at least once, when the signal peptide is removed, and additional processing occurs in many cases, such as prohormone activation. Also, intracellular proteins released into the blood may be processed by proteases, and this processing may be indicative of physiological status of cells in the body;for example, caspase-cleaved proteins in the blood may serve as markers of apoptosis. We have developed a method to profile proteolytic processing in human serum or plasma, based on specific labeling and enrichment of N-termini using an engineered enzyme called subtiligase. After enrichment, N-terminal peptides from proteolytically processed proteins can be identified by LCMSMS. This identification is absolutely depended on sensitive and high-resolution mass spectrometers including the Qstar Elite and Orbitrap instruments in the UCSF Mass Spectrometry Facility. We have identified nearly 1000 unique N-termini in about 300 proteins in whole serum and/or plasma. The identified proteins span nine orders of magnitude in plasma abundance, and include processing sites in complement and coagulation factors, annotated signal sequences, and processing sites in hormones and growth factors, including Gastric Inhibitory Peptide and Vascular Endothelial Growth Factor. Specific proteolytically cleaved peptides are good biomarker candidates and we are currently conducting a pilot study to discover peptide biomarkers of treatment efficacy in patients undergoing chemotherapy for diffuse large B-cell lymphoma (DLBCL).
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