A Mass Spectrometry-Based Proteomics Workflow for Concurrent Profiling of Protein Thiol Oxidation and Phosphorylation.

Multiple types of protein posttranslational modifications (PTMs) play vital roles in the regulation of normal cellular functions and pathogenesis. Two of the most relevant and well-studied PTMs are protein thiol oxidation (redox) and phosphorylation. Both processes involve the reversible addition of a chemical group to a specific amino acid residue, altering the protein activity, stability, or interaction with other molecules. Environmental stressors are known to trigger rapid and dynamic regulation of both thiol oxidation and phosphorylation, and these PTMs on key proteins serve as molecular switches in response to external stimuli. Studies have also shown interplay between phosphorylation and redox modifications, as one PTM type can alter the conformation of a protein, thus exposing or masking the sites for another type of PTM. Such crosstalk represents a complex regulatory mechanism that fine-tunes cellular signaling pathways such as those involved in DNA damage responses (DDR). Despite significant advances in our ability to analyze the redox proteome and phosphoproteome individually, a method that allows the detection of both PTM types from the same sample is still lacking. Herein, we describe a method for simultaneous analysis of protein thiol oxidation and phosphorylation in the same sample. This integrated workflow consists of cell lysis, acetone precipitation, tryptic digestion and isobaric labeling, and subsequent enrichment of thiol-containing peptides utilizing resin-assisted capture (RAC) and phosphopeptides using immobilized metal affinity chromatography (IMAC), respectively. The immediate alkylation of samples and other measures incorporated throughout the protocol prevents artificial oxidation of nascent free thiols and preservation of phosphorylation sites to ensure accurate identification and quantification.