Proteomic Profiling of Oxidative Stress Response Proteins with a Methionine Sulfoxide-Inspired Activity-Based Probe

Abstract

Methionineoxidation is akeyhallmarkof cellular oxidative stress, which is reversed by methionine sulfoxide reductases(Msrs)aspartofcellulardefensemechanisms.Current tools for studyingmethionineoxidationandMsr functionrelyon sulfoxidereductionortranscriptionalanalysis,whichareinadequate formonitoring enzyme activityunder persistent oxidative stress. Moreover, noactivity-basedproteinprofiling(ABPP) toolshave beenreportedfor investigatingthefunctionalstateofMsrsincells. Here, we present SO-acetylene, amethionine sulfoxide-inspired, cysteine-reactive probe for profiling Msrs and other proteins involvedinoxidativestressresponses.Thissubstrate-inspiredprobe preferentiallylabelscatalyticcysteinesofMsrs,servingasanactivity-basedprobe.TheapplicationofSO-acetylenetoEscherichiacoli underhypochlorite stress resulted inthe labelingofmultipleoxidative stress-relatedproteins, revealingdistinct activitypatterns amongMsrs that diverge fromtranscriptional regulation. Furthermore, comparative labelingexperimentswith theconventional probe iodoacetamiderevealedthatSO-acetyleneprovidescomplementarycoverageof cysteinereactivity, efficientlycapturingthe active-sitecysteineof aDJ-1superfamilyglyoxalase,whichispoorly labeledbyiodoacetamide-alkyne.