Detection of reversible protein thiol modifications in tissues

Oxidation/reduction reactions of protein thiol groups (PSH) have been implicated in many physiological and pathological processes. Although many new techniques for separation and identification of modified cysteinyl residues in proteins have been developed, critical assessment of reagents and sample processing often are overlooked. We carefully compared the effectiveness of N-ethylmaleimide (NEM), iodoacetamide (IAM), and iodoacetic acid (IAA) in alkylating protein thiols and found that NEM required less reagent (125 vs. 1000 mol:mol excess), required less time (4 min vs. 4 h), and was more effective at lower pHs (4.3 vs. 8.0) in comparison with IAM and IAA. The relative efficacy of dithiothreitol (DTT) and tris(2-carboxyethyl)phosphine (TCEP) for reducing protein disulfides suspended in NaPO4 buffer or MeOH was assessed, and no differences in total normalized fluorescence were detected at the concentrations tested (10–100 mM); however, individual band resolution appeared better in samples reduced with DTT in MeOH. In addition, we found that oxidation ex vivo was minimized in tissue samples that were homogenized in aqueous buffers containing excess molar quantities of NEM compared with samples homogenized in MeOH containing NEM. Using NEM for thiol alkylation, DTT for disulfide reduction, and mBBr for labeling the reduced disulfide and fluorimetric detection, we were able to generate an in-gel standard curve and quantitate total disulfide contents within biological samples as well as to identify changes in specific protein bands by scanning densitometry. We demonstrated that reagents and techniques we have identified for disulfide detection in complex samples are also applicable to two-dimensional electrophoresis separations.