January 2007 Meeting Announcement, Delaware Valley Mass Spectrometry Discussion Group
- Topic: "Identification of peptidase substrates in human plasma by FTMS based differential mass spectrometry
- Speaker: Nathan A. Yates, Merck Research Laboratories, Rahway NJ 07065
- Date: Monday, January 8, 2007. 6:30 PM
- Time: Social Hour: 6:30 PM.
Talk: 7:30 PM.
Please RSVP to Christopher Petucci PETUCCC@wyeth.com by Thursday January 4th.
- Place: Department of Chemistry, Villanova University (Room 154, Mendel Hall)
Approximately 2% of the human genome encodes for proteases. Unfortunately, however, the biological roles of most of these enzymes remain poorly defined, since the physiological substrates are typically unknown and are difficult to identify using traditional methods. We have developed a proteomics experiment based on FTMS profiling and differential mass spectrometry (dMS) to identify candidate endogenous substrates of proteases using fractionated human plasma as the candidate substrate pool. Here we report proof-of-concept experiments for identifying in vitro substrates of aminopeptidase P2, (APP2) and dipeptidyl peptidase 4 (DPP-4), a peptidase of therapeutic interest for the treatment of type 2 diabetes. For both proteases, previously validated peptide substrates spiked into the human plasma pool were identified. Of note, the differential mass spectrometry experiments also identified novel substrates for each peptidase in the subfraction of human plasma. Targeted MS/MS analysis of these peptides in the complex human plasma pool and manual confirmation of the amino acid sequences led to the identification of these substrates. The novel DPP-4 substrate EPLGRQLTSGP was chemically synthesized and cleavage kinetics were determined in an in vitro DPP-4 enzyme assay. The apparent second order rate constant (kcat/KM) for DPP-4-mediated cleavage was determined to be 2.3 x105 M-1.s-1 confirming that this peptide is efficiently processed by the peptidase in vitro. Collectively, these results demonstrate that differential mass spectrometry has the potential to identify candidate endogenous substrates of target proteases from a human plasma pool. Importantly, knowledge of the endogenous substrates can provide useful insight into the biology of these enzymes and provides useful biomarkers for monitoring their activity in vivo.
Nathan Yates received his undergraduate B.S. degree from Allegheny College in Meadville Pennsylvania and his Ph.D in Analytical Chemistry from the University of Florida in Gainesville Florida. His graduate research focused on the development and application of quadrupole ion trap mass spectrometers for the analysis of complex mixtures. As post-doctoral fellow at the University of Virginia, Nathan coupled an electrospray ionization source to a home built ion trap mass spectrometer and used it to sequence peptides from complex biological samples. He joined Merck Research Laboratories in 1995 and has applied mass spectrometry to to research needs in combinatorial chemistry, medicinal chemistry, drug metabolism, and proteomics. Nathan's current research focuses on the development mass spectrometry based methods for quantitative proteomics. Nathan's mentors include Rick Yost and Jodie Johnson (University of Florida), George Stafford and Mike Story (Thermo Finnigan), Don Hunt and Jeffrey Shabanowitz (University of Virginia), Patrick Griffin (Scripps Florida) and Ron Hendrickson (Merck). He has published fifteen research articles and two patents in the area of mass spectrometry.
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