Analysis of Protein Adduction Kinetics by Quantitative Mass Spectrometry. Competing Adduction Reactions of Glutathione-S-Transferase P1-1 with Electrophiles

Reactive electrophiles formed by xenobiotic metabolism and endogenous oxidative processes covalently modify proteins and these adducts are thought play critical roles both in toxicology and in the pathogenesis of chronic, degenerative diseases [1–3]. Proteomics approaches employing LC-MS-MS together with data analysis algorithms such as Sequest, SALSA and P-Mod have enabled identification of protein adducts [4–7]. MS-MS spectra not only identify the adducted proteins, but also pinpoint the amino acid residue where adduction occurs [5, 6, 8]. Recent application of liquid chromatography-tandem mass spectrometry (LC-MS-MS) proteomics methods and affinity tagged model electrophiles has revealed that protein covalent binding by electrophiles is selective and reproducible, yet highly dependent on the structure and chemistry of the electrophiles [9]. LC-MS-MS methods are also readily used in quantitation of proteins and protein adducts. Relative quantitation is done by incorporating stable isotope labels (2H, 13C, and 15N are most common) [10] into peptide sequences either metabolically [11] or by labeling with small molecule tags [12], which can bind covalently to specific amino acid residues or to peptide N- or C-termini. Our laboratory has described a method for relative quantitation using isotope-labeled phenyl isocyanate (PIC), which labels peptides at the N-terminus [13]. Selectivity of covalent binding governs the biological consequences of adduction. For example, the model electrophiles 1-biotinamido-4-(4′-[maleimidoethylcyclohexane]-carboxamido)butane (BMCC) and biotinyl-iodoacetamidyl-3,6-dioxaoctanediamine (PEO-IAB) both covalently adducted all three subunits of protein phosphatase 2A (PP2A), but only BMCC inhibited PP2A catalytic activity [14]. The inhibition was specifically mapped to adduction of two cysteine residues in the catalytic subunit. Similarly, the model electrophile N-iodoacetyl-N-biotinylhexylenediamine (IAB) and BMCC both adducted the electrophile sensor protein Keap1, but LC-MS-MS analyses revealed different patterns of adduction, which correlated with differential effects on stabilization of the transcription factor Nrf2 and activation of genes regulated by the antioxidant response element (ARE) [15]. A major problem in the analysis of protein adduction is that multiple sites on the same protein could be susceptible to adduction by electrophilic species. Which sites have the highest reactivity? We have adapted a stable isotope tagging method employing N-terminal labeling of peptides with PIC and selective quantiation of the adducted, PIC-labeled peptides by LC-MS-MS. This approach allows the simultaneous measurement of kinetic constants for multiple competing adduction reactions in the same protein. We have used this approach to study the covalent adduction of glutathione-S-transferase P1-1, (GSTP1-1), which has been the subject of previous investigations into the reactivity of cysteine residues and the effects of cysteine modification on GSTP1-1 function [16–19]. This system allowed us to compare the results of kinetic analyses to previously reported structure/function data. Our approach to measuring kinetic reactivity in covalent adduction will be useful for understanding the selectivity and biological consequences of protein modifications involved in chemical toxicity and disease.