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 (PDI) 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 PDI. Human PDI 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 PDI indicates a difference between the redox state of the two active sites. Furthermore, under auto-oxidation conditions an approximately 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 PDI 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 PDI under various conditions.
Kozarova
Anna
A
Department of Chemistry and Biochemistry, University of Windsor, Windsor, Ontario, Canada.
Sliskovic
Inga
I
Mutus
Bulent
B
Simon
Eric S
ES
Andrews
Philip C
PC
Vacratsis
Panayiotis O
PO
D000595:Amino Acid Sequence; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D019704:Protein Disulfide-Isomerases / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D013438:Sulfhydryl Compounds / Q000737:chemistry*
D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D019704:Protein Disulfide-Isomerases; D003545:Cysteine