biostudies-literatureChen SMinisterium f?r Innovation, Wissenschaft und Forschung des Landes Nordrhein-WestfalenChina Scholarship CouncilBundesministerium f?r Bildung und ForschungRegierende B?rgermeister von Berlin?inkl. Wissenschaft und ForschungFonds Wetenschappelijk OnderzoekNational Institutes of HealthMinisterium f?r Kultur und Wissenschaft des Landes Nordrhein-WestfalenNIGMS NIH HHS17512-17520https://www.ebi.ac.uk/biostudies/studies/S-EPMC12416438biostudies-literatureUnknown97(32)Photoaffinity labeling is a common approach in chemical proteomics for the identification of small-molecule probe targets. However, the modification site on the target protein remains difficult to identify because of the probe modification and potential fragmentation during tandem mass spectrometry. We here introduce MS-cleavable photoaffinity groups for a better identification of the modification site. These are based on diazirine photoreactive groups and sulfoxide as the MS-labile linker. We have applied these in photoaffinity probes based on the peptide-like aspartic protease inhibitor pepstatin A, and we demonstrate that we can identify and map binding hotspots on structural models of a target protease.Analytical chemistrySulfoxide-diazirine (SODA) as a Cleavable Photoaffinity Group To Enable the Identification of Photolabeled Modification Sites via LC-MS<sup>3</sup>.PMC12416438201704910881R01GM069832R01 GM069832R35GM152084R35 GM152084G035022NVerhelst SHLSanner MFLoroch SGracias Leone DLChen SForli SfalseSulfoxide-diazirine (SODA) as a Cleavable Photoaffinity Group To Enable the Identification of Photolabeled Modification Sites via LC-MS<sup>3</sup>.Photoaffinity labeling is a common approach in chemical proteomics for the identification of small-molecule probe targets. However, the modification site on the target protein remains difficult to identify because of the probe modification and potential fragmentation during tandem mass spectrometry. We here introduce MS-cleavable photoaffinity groups for a better identification of the modification site. These are based on diazirine photoreactive groups and sulfoxide as the MS-labile linker. We have applied these in photoaffinity probes based on the peptide-like aspartic protease inhibitor pepstatin A, and we demonstrate that we can identify and map binding hotspots on structural models of a target protease.2025-01-01T00:00:00Z2025 Aug2026-06-01T16:08:36.721Z2026-04-08T14:03:45.021ZS-EPMC124164384077068510.1021/acs.analchem.5c02409