A multiplexed targeted mass spectrometric assay for quantifying obesity- associated biomarkers in human plasma.

Background: Obesity is a major global public health challenge that contributes to numerous comorbidities and increased mortality. A better understanding of the biological mechanisms and disease risks associated with obesity requires robust monitoring of key circulating biomarkers, including adipokines, apolipoproteins, and inflammatory proteins. Targeted mass spectrometry (MS) offers a promising platform for developing specific, standardized, and multiplexed assays for biomarker quantification. In this study, we developed a multiplex targeted MS assay for the quantification of 42 obesity-associated biomarker candidates in human plasma. Methods: The assay was optimized for surrogate peptide selection, digestion incubation time, and LC gradient, and evaluated for linearity, lower limit of quantification (LLOQ), imprecision, and stability. A semi-automated sample preparation workflow using 96-well plates was also established to support high-throughput implementation. The assay was applied to a clinical cohort undergoing weight loss interventions to evaluate differences in protein abundance across obesity-related groups and to monitor biomarker changes over time. Statistical analyses were performed to identify proteins with significantly different abundances between study groups and before versus after intervention. Results: The assay demonstrated acceptable linearity, LLOQ, imprecision, and stability. Inter-laboratory validation using samples from 70 healthy individuals showed strong correlation with the finalized standard operating procedure (SOP). Application of the assay to an obesity cohort revealed significant differences in the levels of 6 proteins across obese, overweight, and healthy control groups, as well as 16 proteins that were differentially abundant in obese individuals compared with non-obese individuals (overweight and healthy controls combined). In subjects undergoing weight loss interventions, four proteins-CRP, PRG4, SERPINF1, and SHBG-showed significant concentration changes in individuals who achieved > 5% weight loss. Conclusions: These results demonstrate the robustness and high-throughput capability of this multiplex targeted MS assay for measuring obesity-associated plasma biomarkers. The assay shows potential clinical utility for improving the diagnosis, stratification, and risk assessment of obesity-related conditions, as well as for monitoring responses to weight loss interventions.