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.