{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T09:30:57Z","timestamp":1786527057933,"version":"build-2736575974"},"reference-count":65,"publisher":"MDPI AG","issue":"4","license":[{"start":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T00:00:00Z","timestamp":1786492800000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"name":"King Abdulaziz City for Science and Technology, Saudi Arabia","award":["14-MED-543-02"],"award-info":[{"award-number":["14-MED-543-02"]}]},{"award":["14-MED-543-02"],"award-info":[{"award-number":["14-MED-543-02"]}],"id":[{"id":"https:\/\/ror.org\/05tdz6m39","id-type":"ROR","asserted-by":"publisher"}]}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Methods Protoc.","MPs"],"abstract":"<jats:p>Osteocalcin (OC), a bone protein, is evaluated by its proteoforms, which are implicated in various illnesses. Rapid, clinically used immunoassays are well-established. Epitope dependence, fragment cross-reactivity, uneven identification across carboxylation states, and lack of standardization restrict their efficacy. This study aims to characterize various OC proteoforms in patients with type 2 diabetes mellitus (T2DM) and vitamin D insufficiency and to develop a mass spectrometric immunoassay (MSIA) for their detection. The first MRM-MSIA test to specifically detect OC proteoforms in plasma with vitamin D deficiency-induced alterations in T2DM patients was developed and validated. A precise and selective MRM-MSIA was developed to assess OC fragments in plasma from T2DM patients with normal (DN) or deficient (DD) vitamin D levels. The approach examined OC-1 (aa64\u201371), OC-2 (aa64\u201370), and OC-3 (aa72\u201393) proteoforms. It met international linearity, precision, and accuracy criteria with variability &lt;15% and intra\/inter-day accuracy of 88.8% to 110.1%. The DD group had significantly higher OC-1 levels than the DN group. The receiver operating characteristic (ROC) curve for OC-1 produced an AUC of 0.8263 (95% confidence interval [CI], p = 0.0004). The developed MRM-MSIA measures plasma OC proteoforms with good specificity and sensitivity, making it a promising clinical diagnostic tool.<\/jats:p>","DOI":"10.3390\/mps9040117","type":"journal-article","created":{"date-parts":[[2026,8,12]],"date-time":"2026-08-12T07:57:26Z","timestamp":1786521446000},"page":"117","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Analysis of Osteocalcin Hormone Proteoforms with Vitamin D Deficiency in Patients with Diabetes Mellitus Using Multiple-Reaction Monitoring\u2013Mass Spectrometric Immunoassay Approach"],"prefix":"10.3390","volume":"9","author":[{"ORCID":"https:\/\/orcid.org\/0000-0002-0545-3311","authenticated-orcid":false,"given":"Refat M.","family":"Nimer","sequence":"first","affiliation":[{"name":"Department of Medical Laboratory Sciences, Faculty of Applied Medical Sciences, Jordan University of Science and Technology, Irbid 22110, Jordan"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9745-2959","authenticated-orcid":false,"given":"Hicham","family":"Benabdelkamel","sequence":"additional","affiliation":[{"name":"Proteomics Resource Unit, Obesity Research Center, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8082-9326","authenticated-orcid":false,"given":"Afshan","family":"Masood","sequence":"additional","affiliation":[{"name":"Proteomics Resource Unit, Obesity Research Center, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2244-6842","authenticated-orcid":false,"given":"Maha","family":"Al Mogren","sequence":"additional","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomic Medicine Center of Excellence (GMCoE), King Faisal Specialist Hospital and Research Centre (KFSHRC), Riyadh 11211, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3856-638X","authenticated-orcid":false,"given":"Salini Scaria","family":"Joy","sequence":"additional","affiliation":[{"name":"Strategic Center for Diabetes Research, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9527-9424","authenticated-orcid":false,"given":"Anas M.","family":"Abdel Rahman","sequence":"additional","affiliation":[{"name":"Metabolomics Section, Precision Medicine Laboratory Department, Genomic Medicine Center of Excellence (GMCoE), King Faisal Specialist Hospital and Research Centre (KFSHRC), Riyadh 11211, Saudi Arabia"},{"name":"Department of Biochemistry and Molecular Medicine, College of Medicine, Alfaisal University, Riyadh 11533, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-4792-5188","authenticated-orcid":false,"given":"Assim A.","family":"Alfadda","sequence":"additional","affiliation":[{"name":"Proteomics Resource Unit, Obesity Research Center, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"},{"name":"Strategic Center for Diabetes Research, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"},{"name":"Department of Medicine, College of Medicine, King Saud University, Riyadh 11461, Saudi Arabia"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,12]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","unstructured":"Komori, T. 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