Development and Validation of a Novel LC-MS/MS-Based Proteomics Method for Quantitation of Retinol Binding Protein 4 (RBP4) and Transthyretin (TTR)

Retinol binding protein 4 (RBP4), the circulating carrier of retinol, complexes with transthyretin (TTR) and is a potential biomarker of cardiometabolic disease. However, RBP4 quantitation relies on immunoassays and Western blots without retinol and TTR measurement. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for the simultaneous absolute quantitation of circulating RBP4 and TTR is critical to establishing their biomarker potential. Surrogate peptides with reproducible, linear LC-MS/MS response were selected. Purified proteins were used as quantitation standards and heavy-labeled peptides as internal standards. Matrix effects were evaluated. The validated method was applied to measure inter- and intraindividual variability in RBP4 and TTR concentrations in healthy individuals and patients with diabetic kidney disease. Quantitation was linear for the clinically relevant concentration ranges of RBP4 (0.5–6 μM) and TTR (5.8–69 μM). Assay interday variability was <12% and precision within 5%. The interindividual variability for RBP4 and TTR concentrations was 18–26%, while intraindividual variability was similar to assay variability. RBP4 and TTR quantitation correlated with commercially available enzyme-linked immunoassays (ELISA) assays. The developed LC-MS/MS method enables simultaneous absolute quantitation of RBP4 and TTR in serum and plasma. It can be applied to clinical biomarker studies and stoichiometric measurements of circulating RBP4, TTR, and retinol.