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High-Speed Proteomics

presented by

Steven J. Bark
The Scripps Research Institute

November 30, 2000

The Scripps Research Institute, W.M. Keck Foundation Amphitheater


Background:

Looking at Steve Bark's scientific career, even at this relatively early stage, his conviction towards Scripps is clear. After graduating magna cum laude with a B.S. in Chemistry from U.C. Riverside in 1991, Steve came to The Scripps Research Institute to pursue graduate study. His first couple years were spent researching the design and synthesis of selective therapeutic agents for cancer chemotherapy under Darryl C. Rideout. Steve's Ph.D. thesis work on "The Chemical Ligation of Unprotected Peptide Segments Using Auxiliary Functional Groups" was conducted from 1994 to 1997 under the advisement of Stephen B. H. Kent. After receiving his Ph.D. in Macromolecular & Cellular Structure and Chemistry, Steve completed an NRSA postdoctoral fellowship at TSRI and in January, 2000 became the Manager of the Open Access Mass Spec Facility.

Abstract:

Protein mass mapping using proteolytic digestion has become a standard method for characterizing and identifying proteins. Two limitations of this method for automated high-throughput analysis are the time required for adequate proteolysis and the resistance of some proteins to digestion. Digestion with trypsin typically requires 4 to 24 hours and many compact globular proteins exhibit resistance to cleavage and cannot be effectively analyzed by available procedures. The key to our method is the use of a thermophilic enzyme, thermolysin, with optimal activity at elevated temperatures. Thermolysin has not been previously utilized for protein identification because of apparent low specificity. However, our MALDI and LC/MS/MS experiments indicate that thermolysin does have specificity and that this specificity is not an impediment to accurate protein identification. The predominant cleavage site residues for this protease have been defined by our experiments. Examples of identification will be discussed.

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