Proteomics Platform Meeting
October 12, 2001
Leiden, The Netherlands
Targeted Proteomics in Molecular Biology.
Paul Tempst, Ph.D.
Professor of Molecular Biology
Memorial Sloan-Kettering Cancer Center
and Cornell University (Weill Medical Campus)
New York, NY
Proteomics is the new new thing. The global variety aims to map expression of all proteins in all cells at any given point, by using 2D-gels and high-throughput mass spectrometry. This is the protein equivalent of DNA arrays. A complementary, focused embodiment of proteomics, termed targeted, calls for the examination of subsets of the proteome, e.g. those proteins that bind to particular DNA sequences, to other proteins or small molecules, or exist as members of higher order complexes, or any combination thereof. We seek to apply this concept to the study of transcriptional regulation and of other cellular processes.
In addition, precise occurrence and sites of post-translational protein modifications cannot be easily inferred from genomic analyses either. Cataloging modified proteins, taken in the proper biological context, is a strictly biochemical problem; and an important one. For instance, reversible phosphorylation, just one of many examples, is critical for transmission of signals in all living cells. We are exploring novel approaches for micro-scale capture of modified proteins, both for identification and subsequent mapping of the modified amino acids.
It is imperative that low abundant proteins and proteins that can only be isolated from limited source material are an integral part of these studies. Most current protocols for protein purification / display and for automated identification yield rather low recoveries, thus limiting the overall process in terms of sensitivity and speed (e.g. by having to collect and process more starting material).
In my presentation, I will describe our R&D efforts that focus on four areas: (i) selective micro-capture of target protein complexes and of specifically modified proteins; (ii) optimization of sample handling steps leading to mass analysis of fragments, in submicroliter volumes; (iii) multi-platform MS based protein identifications; and (iv) development of a Protein Interactions in the Nucleus database (PINdb).