| Method |
Differential labeling of protein thiols and disulfides, followed by chromatographic or electrophoretic separation of cellular proteins. Then, cysteine containing proteins are identified by mass spectrometry and in some cases information of the thiol–disulfide status of peptides are also determined. Cells from a 95% confluent 10-cm dish were harvested by trypsinization, washed in PBS, and directly quenched by addition of 10% TCA.Total GS were quantified by reducing disulfides with TCEP followed by thiol derivatization with NPM. For quantification of GSSG, free thiols were first blocked by NEM followed by TCEP reduction and NPM derivatization. GSH equivalents were calculated by subtracting GS in GSSG from total GS. |