Identification of redox sensitive thiols of protein disulfide isomerase using isotope coded affinity technology and mass spectrometry

Regulation of the redox state of protein disulfide isomerase (PDT) is critical for its various catalytic functions. Here we describe a procedure utilizing isotope-coded affinity tag (ICAT) technology and mass spectrometry that quantitates relative changes in the dynamic thiol and disulfide states of human PDT. Human PDT contains six cysteine residues, four present in two active sites within the a and a' domains, and two present in the b' domain. ICAT labeling of human PDT indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an similar to 80% decrease in available thiols within the a domain was detected. Surprisingly, the redox state of one of the two cysteines, Cys-295, within the b' domain was altered between the fully reduced and the auto-oxidized state of PDT while the other b' domain cysteine remained fully reduced. An interesting mono- and dioxidation modification of an invariable tryptophan residue, Trp-35, within the active site was also mapped by tandem mass spectrometry. Our findings indicate that ICAT methodology in conjunction with mass spectrometry represents a powerful tool to monitor changes in the redox state of individual cysteine residues within PDT under various conditions. (c) 2007 American Society for Mass Spectrometry.