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 imagi...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.展开更多
基金supported by the National Key Research and Development Program of China(grant no.2022YFC3400800)the National Natural Science Foundation of China(grant nos.22434001 and 82121004)+1 种基金the China Postdoctoral Science Foundation(grant no.2022M710734)the China Postdoctoral Science Foundation for Innovative Talents(grant no.BX20220076).
文摘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.
