Protein unfolding, disassembly, and aggregation underlie many diseases, but proteomic scale study of these processes has been limited. Cysteine Shotgun labeling of cells and complex lysates can be done efficiently with fluorescent dyes that survive Mass Spectrometry fragmentation.  Here we study whole-cell lysates in native versus urea-denaturing conditions and in live cells all as a function of time and temperature (20-45 deg-C). Labeling rate constants are calculated for any given Cys site by normalizing the protein labeling kinetics to the rapid labeling under denaturing conditions. Fluorescence bands in SDS-PAGE provide a first measure of labeling, but MS identifies proteins and pinpoints specific, susceptible sites. Double-labeling schemes and live cell labeling take advantage of the varied solubility of dyes such as monobromobimane. A number of proteins, many of them abundant cytoskeletal proteins implicated in disease, contain cys with a wide variety of rate constants and appear especially amenable to study. Many of the targeted cysteines are near sites of phosphorylation and mutations, creating an important new source of data on the effect of these key sites.

Kinetic labeling for cysteine shotgun mass spectrometry (CS-MS) maps the foldome

244th National Meeting (2012)

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