JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST004097/files/direct.pg_matrix.tsvhttps://storage.jpostdb.org/JPST004097/files/filter_report.tsvhttps://storage.jpostdb.org/JPST004097/files/direct_report.tsvhttps://storage.jpostdb.org/JPST004097/files/direct.pr_matrix.tsvhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_4-1.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_4-3.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-8_Temp4_stop-4.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-8_Temp4_stop-2.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-7_Temp4-2.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-7_Temp4-4.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_3-2.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_3-4.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_4-4.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_4-2.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-7_Temp4-1.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-8_Temp4_stop-3.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_3-3.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-8_Temp4_stop-1.rawhttps://storage.jpostdb.org/JPST004097/files/240524_miya-7_Temp4-3.rawhttps://storage.jpostdb.org/JPST004097/files/miya_LIP-MS_3-1.rawprimary200OKProteomicsYusuke KawashimaHomo Sapiens (human)Cellular Organismshttps://repository.jpostdb.org/entry/JPST004097Kitasato UniversityjPOSTLimited proteolysis mass spectrometry (LiP-MS) is a powerful approach for probing protein conformational changes on a proteome-wide scale. However, conventional workflows rely on a two-step digestion with proteinase K and trypsin, which increases complexity and reduces reproducibility and sensitivity. This study aimed to develop a simplified one-step protocol, termed Swift Trypsin LiP-MS (STLiP-MS), which uses a trypsin-immobilized spin column and high-speed centrifugation to achieve rapid and reproducible surface-limited proteolysis. Using HEK293 cell extracts, STLiP-MS identified 286 proteins exhibiting conformational changes upon phosphatase inhibition, including 37 enriched in phosphatase-related Gene Ontology categories. The method improvements, including suppression of predigestion and immediate enzyme inactivation, further increased sensitivity, enabling the detection of 799 proteins with structural alterations, of which 77 were enriched in phosphatase-related categories. Comparison with the single-pot solid-phase-enhanced sample preparation (SP3) method confirmed that these changes originated from structure-selective proteolysis and were not detectable under fully denaturing conditions. To demonstrate its broader applicability, we applied STLiP-MS to the adenosine A_2A receptor (A_2A-BRIL) and observed antibody-induced protection of extracellular loop 2 (residues 147-176). Cryogenic electron microscopy validated Fab fragment binding to the same region, confirming the correspondence between STLiP-MS signals and actual antibody-antigen interfaces. Collectively, these results show that STLiP-MS is a rapid and robust platform that enables sensitive, label-free detection of local structural changes under near-physiological conditions and accurate prediction of protein-protein interaction sites. This method holds great promise for applications in structural proteomics and drug target identification.Rapid and Label-Free Structural Proteomics Using One-Step Swift Trypsin LiP-MS.Miyashita Yasuomi Y, Konno Ryo R, Ogasawara Satoshi S, Okuda Yusei Y, Takamuku Yuuki Y, Moriya Toshio T, Saito Tetsuichiro T, Murata Takeshi T, Ohara Osamu O, Kawashima Yusuke YfalseRapid and Label-Free Structural Proteomics by One-Step Swift Trypsin LiP-MSWe developed a rapid, surface-targeted limited proteolysis workflow, Swift Trypsin LiP-MS (STLiP-MS), to profile proteome-wide structural changes under near-native conditions. STLiP-MS uses a trypsin-immobilized spin column and centrifugation-controlled contact to achieve instantaneous, highly reproducible limited digestion, followed by reduction/alkylation and desalting. Proof-of-concept analyses were performed on HEK293T cell lysates (± phosphatase inhibitor PhosSTOP) and on a purified GPCR complex (A2A-BRIL with IgG). Peptides were separated on a 75-µm ID C18 column (50 °C) using an UltiMate 3000 RSLCnano and analyzed on an Orbitrap HF-X. Both DDA (top-50 HCD, 70-min gradient) and DIA (variable windows, 70-min gradient) runs were acquired. DIA data were processed with DIA-NN (library-free/in-silico; UniProt human, 2024-03), and DDA of purified samples with PEAKS Studio 12.5.Mon Jan 05 00:00:00 GMT 2026PXD069771131567960641552556