Cysteine is a relatively hydrophobic amino acid and is often buried within protein folds or assemblies, but it is rarely used to probe protein structure. For intracellular proteins, Cys are not tied up in disulfides, and so their reactivity to many fluorescent dyes in solution is readily determined as a function of temperature under both native and high urea conditions. When a protein is folded at low temperature, the reaction rate tends to be faster in urea, and the ratio of kinetics yields a thermal unfolding curve that appears very similar to results from other solution methods, such as CD. Unlike these other methods, however, the Cys labeling approach can also be applied to intact cells with membrane-permeable, cell-viable dyes at low dose. Labeling studies of stem cells, lung cancer cells, muscle cells, and additional cells treated under two different conditions, such as adherent or trypsin-released, are followed by electrophoretic separation and detailed LC/MS-MS study. What results is an identification of labeled proteins as well as labeled sites in many proteins, including disease related structural proteins such as Lamin-A/C and Dystrophin. The Cys shotgun method thus provides unique, proteomic scale measurements of protein structural changes in both solution and intact cells.

(Science 317, 663-666, 2007)