Site-specific proteomic mapping of modified cysteine residues

Abstract

The wide reactivity of the thiol group enables the formation of a number of chemically and biologically distinct posttranslational modifications. Proteins within nearly all major families undergo some form of cysteine modification and the modifications are associated with regulatory functions across many biological processes. However, the susceptibility of thiols to redox shifts, as well as the labile nature of most thiol modifications, renders detection difficult. Analysis difficulties are compounded further in complex protein mixtures due to the typical low abundance of cysteine modifications under normal physiological conditions. Here we describe methods for the analysis of three cysteine modifications: Nitrosylation, glutathionylation, and S-acylation. The three methods use the same organic mercury-conjugated agarose resin as an enrichment platform. To date, over 2154 sites on 1446 proteins have been identified between the three modifications using this method. Using equivalent processing, enrichment, and analytical methods has enabled a more comprehensive picture of the redox proteome landscape.