Title: BEYOND GENOMICS

Abstract

The genomics rush of the past two decades based on DNA sequencing is now reaching maturity. It is time to move on to what cells do with this information. Some questions of great interest are which genes express proteins, when do they do so, the conditions under which expression occurs, and how does regulation of specific proteins occur. This has lead to efforts to define the proteome (i.e., to catalog all expressed proteins in a cell). Recent discussions in the popular press indicate that some companies are thinking of cataloging proteomes in huge analytical factories using thousands of 2-D gel electrophoresis systems, hundreds of robots for spot excision from gels, huge numbers of trypsin digestors, dozens of mass spectrometers, many high speed computers and armies of people to run them.

This talk will begin by addressing the question of how regulation occurs in cells, where and why genomics could fail to reveal how cells are regulated, the extent to which protein diversity must be addressed in examining cellular regulation, discuss the issue of high throughput proteomics, and examine the need for quantification in recognizing small numbers of proteins that respond to external stimuli.

Efforts to simplify proteomics and address quantification issues will be presented describing a multidimensional chromatographic approach with signature peptides. The premise in this approach is that tryptic peptides can be selected from complex mixtures, resolved by reversed phase chromatography, and be used as analytical surrogates for the protein from which they were derived. It will be shown that i) it is easier to separate and identify signature peptides than intact proteins in many cases, ii) proteins do not have to be soluble for identification, and iii) it is easier to tryptic digest all proteins in a single reaction than to isolate and digest each individually as in the 2-D electrophoresis approach. Finally, a heavy isotope labeling strategy will be described from quantification.