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

BACKGROUND

Retinol binding protein 4 (RBP4) is the plasma carrier of retinol that complexes with transthyretin (TTR). RBP4 is a potential biomarker of cardiometabolic disease. However, RBP4 quantitation has relied on immunoassays and western blots without concurrent measurement of retinol and TTR. A liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous absolute quantitation of serum and plasma RBP4 and TTR is needed to advance the understanding of RBP4 and TTR as biomarkers.

METHODS

Surrogate peptides with reproducible, linear LC-MS/MS response were selected for RBP4 and TTR quantitation. Purified proteins were used as quantitation standards and heavy labelled peptides as internal standards. Matrix effects were evaluated for quantitation. The method was validated using pooled human serum and applied to measure inter- and intra-individual variability in RBP4 and TTR concentrations in healthy individuals and in patients with diabetic kidney disease.

RESULTS

The quantitation was linear for the clinically relevant concentration ranges of RBP4 (0.5 - 6 μM) and TTR (5.8 - 69 μM). The assay inter-day variability was <12% and precision within 5%. The inter-individual variability for RBP4 and TTR concentrations was 18 - 26%, while intra-individual variability was similar to assay variability. RBP4 and TTR quantitation correlated with commercially available ELISA assays.

CONCLUSIONS

The developed LC-MS/MS method allows simultaneous absolute quantitation of RBP4 and TTR in human serum and plasma with reduced samples volumes. The method can be applied to clinical biomarker studies, for analysis of nutritional vitamin A status, and measurements of stoichiometry of RBP4, TTR and retinol in circulation.