April 2004 Meeting Announcement, Delaware Valley Mass Spectrometry Discussion Group
- Topic: "Proteomics: How Many Proteins Can We Identify? Can We Do Better? Are We Sure About Those Identities?
- Speaker:Ron Orlando, Complex Carbohydrate Research Center, University of Georgia
- Date: Monday, April 12, 2004. 6:30 PM
- Time: Social Hour: 6:30 PM. (Pizza)
Talk: 7:30 PM.
- Place: Widener University, Webb Room.
The typical "gel-less" proteomics approach involves early proteolytic digestion of the proteome, followed by two-dimensional LC coupled with MS/MS. Inspection of the data we obtained using this two-dimensional LC approach on a range of complex proteomes, revealed that only one out of five peptides, with sufficient intensity to provide amino acid sequence, were actually subjected to MS/MS analysis. The reason for this is that too many peptides co-elute leaving the mass spectrometer insufficient time to analyze the majority of them. This observation led us to the conclusion that additional separation steps were needed to increase the coverage of complex proteomes. Our first attempt at increasing proteome coverage consisted of adding an additional RP separation prior to the standard SCX/RP. We have used this three-dimensional LC-MS/MS approach for the analysis of the Trypanosoma cruzi proteome in its epimastigote stage. T. Cruzi is of interest since it is the intracellular protozoan parasite that causes Chagas disease, -- the principle cause of mortality in Latin America. In our first attempt, we obtained over 25,000 peptide sequences, which identified greater than 35% of the proteins in the partial T. cruzi genome, despite analyzing only one of the four stages in this organisms life cycle. In addition, we were able to identify over 900 proteins from the bovine serum proteome, which was used as the growth media for the parasite. These numbers are staggering since the best current serum proteome reported identified 33% fewer proteins than observed in this study, where the serum proteins were considered a contaminant and multiple procedures were used to remove them from the parasites. This presentation will also include discussion on approaches to provide more complete coverage and issues related to dynamic range, our methods to evaluate the confidence in protein identification, as well as description of time saving procedures.
- Bio:Ron Orland received a BS in Natural Science from St. Mary’s College of Maryland in
1983, and Ph.D. in Analytical Chemistry from the University of Delaware in 1988, under the advisement of Dr. Burnaby Munson. Prior to joining the Complex Carbohydrate Research Center (CCRC) at the University of Georgia in 1993, Ron spent three years at the Structural Biochemistry Center at the University of Maryland Baltimore County and two years at the Suntory Institute for Bioorganic Research in Osaka, Japan. He is now an Associate Professor at the CCRC and a Faculty Member of The Plant Center at UGA. In addition, Ron is an Associate Research Scientist with the Ovarian Cancer Institute in Atlanta, and a Science Advisory Board Member of 1st EnviroSafety, Inc. in St. James City, Florida.
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