Gross Abstract

Michael L. Gross
Department of Chemistry
Washington University

"Mass Spectrometry-Based Protein Footprinting: The Fourth Pillar of Proteomics"

Abstract:

A variety of chemical modifications have been developed over the years to footprint proteins, to determine interfaces and sites of ligand interaction and to understand protein folding We are interested in implementing these procedures in protein mass spectrometry. One can view these protein tools as the fourth pillar of mass-spectrometry based proteomics. Three pillars are identifying proteins from mass and sequence tags, quantifying them, and determining post-translational modifications. The motivation to establish a fourth pillar is to use the high sensitivity of mass spectrometry to learn biophysical properties of proteins.

In the area of H/D exchange, we use a recently developed tool of PLIMSTEX (protein ligand interactions by mass spectrometry, titration, and H/D exchange) to determine binding affinity and to locate binding regions between a protein and ligand. Complementing H/D exchange are highly specific reactions such as acetylation and iodination as footprinting procedures. We have used both to understand protein/DNA binding. An intermediate strategy is OH radical footprinting, which we have implemented in a fast experiment to label aromatic, some aliphatic, and sulfur-containing amino acids in proteins. We have strong evidence that this approach to footprinting is indeed faster than the that fastest early steps of protein unfolding.

These and other approaches involving chemical reactivity are enabled by the powerful analytical proteomic strategies of modern mass spectrometry.