The effect of Trx-1 overexpression on protein expression and redox state of cysteines by iodoTMT and TMT labeling

Abstract

ADAM17 is a membrane metalloproteinase which has the main function of modulating cell surface signaling event by releasing surface protein ectodomains making it soluble to interact with cellular receptors. The thioredoxin system is a major disulfide reductase system critical for DNA synthesis and defense against to oxidative stress. Several studies show the modulation of ADAM17 activity and our group identified Trx-1 as interaction partner. One of our recent findings is that ADAM17 favors the monomeric form of Trx-1 and it appears to be associated with Trx-1 activity. The constant and growing need for quantitative measurement of relative protein amounts methods, allowed the introduction of different strategies involving isotopes label such as iodoTMT and TMT. The iodoTMT reagents are sets of isobaric (mass and structure) isomers that are iodoacetyl-activated for covalent, irreversible labeling of sulfhydryl (-SH) groups. Similar to iodoacetamide, iodoTMT reagents react specifically with reduced cysteines in peptides and proteins being differentiated by mass spectrometry, enabling quantitation of the relative abundance of cysteine modifications, such as S-nitrosylation, oxidation and disulfide bonds, in cultured cells grown or treated with different conditions. The isobaric tagging, as TMT, is achieved by using chemical moieties or tags which are identical in mass, so that all derivatised peptides are isobaric, chromatographically indistinguishable and yielding a single peak in the mass spectrum for both samples (normal and sick samples). However, the relative abundances of the isobarically tagged peptides are revealed when the moieties fragment during MS/MS experiments to release reporter ions with different masses. TMT contain a reactive, a normalizer and a reporter groups. As one of our findings is that the monomeric form of Trx-1 by ADAM17 appears to be associated to Trx-1 activity, the aim of this project is to analyze the redox state of total cysteines in cell lysate by iodoTMT and the total protein expression by TMT with Trx-1 and mutant and wildtype cytoplasmic domain of ADAM17.