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In the absence of Ca2+, no detectable CDZ binding was observed. However, CDZ binding was observed when calmodulin was fully loaded with Ca2+. CDZ binding to the protein caused marked redistribution of the CSD toward lower charge states, consistent with ligand-induced stabilization of the protein into a more compact conformational ensemble. The apparent dissociation constant (Kd) of the interaction was determined to be 261 \u00b1 29 nM and 126 \u00b1 17 nM from Langmuir and quadratic binding models, respectively. Complementary in-droplet HDX-MS revealed that deuterium uptake was correlated with charge state across the CSD, corroborating the nMS evidence for stepwise conformational compaction of CaM. Within the individual charge states, two distinct populations representing CDZ-bound and unbound CaM were resolved by HDX-MS, which were not detectable by nMS alone. Comparison of these populations within each of the 6+, 5+ and 4+ charge states revealed reductions in deuterium uptake by approximately 37%, 22%, and 11% upon CDZ binding, respectively, consistent with ligand occlusion and local structural tightening at the binding interface. The gradient in protection across charge states further reflects differences in binding interface accessibility among CaM conformers spanning a range of CDZ-induced conformational states. Together, these results demonstrate that cVSSI-based nMS coupled with in-droplet HDX-MS provides an integrated platform for simultaneously resolving metal loading, ligand binding, binding affinity, and ligand-induced conformational changes. This approach complements traditional structural methods by enabling direct interrogation of dynamic, metal-dependent protein\u2013ligand interactions in their native states.<\/jats:p>","DOI":"10.1021\/acsomega.6c07079","type":"journal-article","created":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T03:27:17Z","timestamp":1788406037000},"page":"54578-54592","source":"Crossref","is-referenced-by-count":0,"title":["Structural Characterization\nof Calcium-Dependent Calmodulin-Calmidazolium\nBinding Using Capillary Vibrating Sharp-Edge Spray Ionization-Based\nNative Mass Spectrometry and In-Droplet Hydrogen\u2013Deuterium\nExchange Mass Spectrometry"],"prefix":"10.1021","volume":"11","author":[{"given":"Sohag","family":"Ahmed","sequence":"first","affiliation":[{"name":"West Virginia University , , , ,","place":["Morgantown, West Virginia, United States, 26506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-6900-944X","authenticated-orcid":true,"given":"Axel","family":"Woehrling","sequence":"additional","affiliation":[{"name":"West Virginia University , , , ,","place":["Morgantown, West Virginia, United States, 26506"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peng","family":"Li","sequence":"additional","affiliation":[{"name":"Lehigh University , , 6 East Packer Avenue , , ,","place":["Bethlehem, Pennsylvania, United States, 18015"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4360-2928","authenticated-orcid":true,"given":"Stephen J.","family":"Valentine","sequence":"additional","affiliation":[{"name":"West Virginia University , , , ,","place":["Morgantown, West Virginia, United States, 26506"]},{"name":"University of Louisville , , , ,","place":["Louisville, Kentucky, United States, 40292"]}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1315-4917","authenticated-orcid":true,"given":"Kevin C.","family":"Courtney","sequence":"additional","affiliation":[{"name":"West Virginia University , , , ,","place":["Morgantown, West Virginia, United States, 26506"]},{"name":"University of Guelph , , , ,","place":["Guelph, Ontario, Canada, N1G 2W1"]}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"316","published-online":{"date-parts":[[2026,8,31]]},"reference":[{"issue":"5","key":"2026091510082558000_cit1","doi-asserted-by":"publisher","first-page":"457","DOI":"10.3390\/membranes13050457","article-title":"Advances in Mass Spectrometry on\nMembrane Proteins","volume":"13","author":"Yang","year":"2023","journal-title":"Membranes"},{"issue":"4","key":"2026091510082558000_cit2","doi-asserted-by":"publisher","DOI":"10.1002\/pro.4612","article-title":"Dissecting\nthe Structural Heterogeneity\nof Proteins by Native Mass Spectrometry","volume":"32","author":"Reid","year":"2023","journal-title":"Protein\nSci."},{"issue":"18","key":"2026091510082558000_cit3","doi-asserted-by":"publisher","first-page":"3897","DOI":"10.1007\/s00216-023-04584-z","article-title":"Emergence of Mass Spectrometry Detergents for Membrane\nProteomics","volume":"415","author":"Behnke","year":"2023","journal-title":"Anal. 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