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Here, we introduce aziridination-based lipid\u2013protein crosslinking coupled with mass spectrometry (Azi-LPXL-MS), a chemoselective strategy that installs an N\u2013H aziridine moiety onto acyl chains of native unsaturated lipids and enables subsequent crosslinking to proximal lysine residues using the amine-reactive reagent disuccinimidyl suberate (DSS). This integrated workflow simultaneously identifies lipid-binding sites and intraprotein peptide\u2013peptide distance constraints, providing direct insight into lipid binding and binding-induced conformational changes within a single experiment. Application of Azi-LPXL-MS to \u03b2-lactoglobulin (BLG) demonstrates these capabilities, consistent with known structural models, while additionally providing solution-phase and conformational information not accessible from static structures alone. The power of the method is further demonstrated in the integral membrane enzyme diacylglycerol acyltransferase 1 (DGAT1). Azi-LPXL-MS reveals phosphatidic acid (PA)-binding sites and maps lipid-dependent conformational rearrangements across both resolved and previously uncharacterized regions. These data further define an allosteric conformational network linking lipid engagement to distal structural changes. Integrated with site-directed mutagenesis and molecular docking, two potential PA-binding sites in DGAT1, located in a surface pocket and an internal chamber, were identified and functionally validated, further supporting the experimental findings. These results establish Azi-LPXL-MS as a site-resolved method for studying lipid\u2013protein interactions, providing insight into how individual lipid species engage proteins and influence their conformations in biological systems.<\/jats:p>","DOI":"10.1021\/jacs.6c08027","type":"journal-article","created":{"date-parts":[[2026,10,7]],"date-time":"2026-10-07T21:01:47Z","timestamp":1791406907000},"page":"44070-44078","source":"Crossref","is-referenced-by-count":0,"title":["Aziridination-Based\nCrosslinking Enables Site-Resolved\nElucidation of Lipid Binding and Protein Conformational Changes"],"prefix":"10.1021","volume":"148","author":[{"ORCID":"https:\/\/orcid.org\/0009-0008-7901-7142","authenticated-orcid":true,"given":"Chia-Lung","family":"Tsai","sequence":"first","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-7802-3948","authenticated-orcid":true,"given":"Atiya Tahira","family":"Tasnim","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 300 Olsen Boulevard , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhenyu","family":"Xi","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Syuan-Ting","family":"Kuo","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gopal Reddy","family":"Ramidi","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Jiaxin","family":"Feng","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Michael Ryan","family":"Eastabrook","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0830-3914","authenticated-orcid":true,"given":"David H.","family":"Russell","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xuewu","family":"Sui","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 300 Olsen Boulevard , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-8292-130X","authenticated-orcid":true,"given":"Xin","family":"Yan","sequence":"additional","affiliation":[{"name":"Texas A&M University , , 580 Ross Street , , ,","place":["College Station, Texas, United States, 77843"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,10,7]]},"reference":[{"issue":"12","key":"2026102111062873200_cit1","doi-asserted-by":"publisher","first-page":"753","DOI":"10.1038\/nrm4080","article-title":"The systematic analysis of protein\u2013lipid\ninteractions comes of age","volume":"16","author":"Saliba","year":"2015","journal-title":"Nat. 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Rev. Biochem."},{"issue":"38","key":"2026102111062873200_cit26","doi-asserted-by":"publisher","first-page":"20859","DOI":"10.1021\/jacs.3c05883","article-title":"Identifying Membrane\nProtein\u2013Lipid Interactions with Lipidomic Lipid Exchange-Mass\nSpectrometry","volume":"145","author":"Zhang","year":"2023","journal-title":"J. Am. Chem. Soc."},{"issue":"25","key":"2026102111062873200_cit27","doi-asserted-by":"publisher","first-page":"10426","DOI":"10.1021\/acs.analchem.4c01704","article-title":"Simultaneous Native\nMass Spectrometry Analysis of Single and Double Mutants To Probe Lipid\nBinding to Membrane Proteins","volume":"96","author":"Jayasekera","year":"2024","journal-title":"Anal. 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