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) is the plasma carrier of retinol that complexes with transthyretin (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. 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.
The assay inter-day variability was <12% and precision within 5%. RBP4 and TTR quantitation correlated with commercially available ELISA assays.