Characterization of S‐thiolation on secreted proteins fromE. coliby mass spectrometry

Abstract

S-thiolation is a reversible post-translational modification in which thiol metabolites of low molecular masses are linked to protein sulfhydryl groups through disulfide bonds. This modification is commonly observed in recombinant proteins secreted from E. coli cells. Since it can alter protein functions and introduce molecular heterogeneity, S-thiolation is undesirable for recombinant protein production. To date, few published studies have characterized thiol modifiers or investigated the mechanism of S-thiolation in recombinant proteins. In this work, reversed-phase liquid chromatography and mass spectrometry were used to characterize four of the most abundant thiol modifiers on recombinant proteins secreted from E. coli BL21 (DE3) strain. These thiol modifiers have been identified as glutathione, 4-phosphopantetheine, gluconoylated glutathione, and dephosphorylated coenzyme A. S-thiolation by these thiol modifiers increases protein mass by 305, 356, 483, and 685 Da, respectively. These specific mass increases can be used as markers for identifying S-thiolation in recombinant proteins.

Mass spectrometry in studies of protein thiol chemistry and signaling: Opportunities and caveats

“…The characteristic Δ mass signals for S -cysteinylation, S -homocysteinylation, and S -glutathionylation from the parent protein are + 119, +143, and + 305 Da, respectively. To identify the type of S -thiolation, a control experiment may be performed in which the sample is treated with DTT followed by MS analysis to monitor the loss of mass corresponding to either Cys, Hcy, and GSH [96, 97]. Recent advances in MS approaches, like top-down proteomics, allow for the analysis of S -thiolation in intact single proteins and simple mixtures and have been recently demonstrated with both protein S -glutathionylation and S -cysteinylation [98, 99].…”