One-step labeling with a fluorinated porphyrin-based probe enables in situ imaging and profiling of the thiol proteome

Profiling the thiol proteome in live cells is a critical yet challenging task for elucidating oxidative stress-related processes due to the scarcity of multifunctional probes that directly integrate fluorescence imaging with chemical proteomics. Here, we constructed a fluorescent, enrichable, and mass spectrometry-compatible probe based on fluorinated porphyrin, termed the FP probe. By eliminating the need for attaching separate reporter modules after probe labeling, the FP probe not only directly visualizes the thiol proteome in cells but also enables the visualization of thiol proteome enrichment for reliable site identification by mass spectrometry. This capability drives the development of a visualization-guided proteomics (VGP) workflow, which seamlessly merges fluorescence imaging with chemical proteomics. In addition, the FP probe demonstrated an advantage in the capture of cysteines with low solvent accessibility. We successfully identified Cys818 of AP2B1, a residue with a relative solvent accessible area of 0.02 that is sensitive to the protein interactions induced by diamide stress in live cells. Our probe may pave the way for the design of multifunctional probes and become an important tool for applications including target identification, drug discovery, and diagnostics.