Analysis of Osteocalcin Hormone Proteoforms with Vitamin D Deficiency in Patients with Diabetes Mellitus Using Multiple-Reaction Monitoring–Mass Spectrometric Immunoassay Approach

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 (aa64–71), OC-2 (aa64–70), and OC-3 (aa72–93) proteoforms. It met international linearity, precision, and accuracy criteria with variability <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.