Chemical Probe Development for Omics Analysis

Abstract

Oxidative stress generates diverse protein and metabolite modifications critical to redox signaling and disease pathology, yet many of these modifications remain poorly characterized at the omics level. The primary obstacle has been the lack of chemical tools capable of detecting these modifications in complex biological systems. This thesis addresses this gap through the development of chemical probes targeting three previously inaccessible oxidative modifications: methionine sulfoxide repair systems, thiyl radical formation, and endogenous aldehyde species. SO-acetylene, a methionine sulfoxide-mimicking probe, enables activity-based profiling of methionine sulfoxide reductases (Msrs) by directly labeling catalytic cysteine residues. This activity- dependent labeling reveals substantial divergence between transcriptional regulation and actual enzyme activity during oxidative stress, which information is inaccessible through conventional fluorescence- based or transcriptional approaches. Comparative proteomics further demonstrates that SO-acetylene efficiently captures DJ-1 superfamily proteins and aldehyde reductases, complementing the cysteine reactivity profile of traditional iodoacetamide-based probes. Thiyl radicals pose a distinct detection challenge due to their transient nature and high reactivity. We developed thioacetylene probes that trap these radical species through enhanced radical-mediated thiol- yne chemistry, enabling proteome-wide characterization with site-specificity which is limited by using traditional EPR spectroscopy or spin-trapping methods. These probes successfully detected thiyl radical formation in purified proteins and E. coli cells under superoxide-induced stress. Preliminary work with TMT-alkoxyamine demonstrates improved labeling of cellular aldehyde metabolites for mass spectrometry-based chemo-metabolomics, addressing detection limitations of non-polarized aldehyde species. With isotope-labeling, quantitative chemo-metabolomics are available, which contributes to studying the cellular aldehyde metabolisms. Beyond oxidative modifications, this thesis presents phosphoarginine analogs that identify phosphoarginine-binding proteins including YwlE phosphatase, -glucosidase BglH, and several chaperones. We also show that amidine structures function as glucosidase and glucuronidase inhibitors, with potential applications in gut microbiome research. This work expands the chemical toolbox for interrogating oxidative stress at the proteomic level while demonstrating broader applicability of chemical probe strategies to diverse biological questions.