A fully validated liquid chromatography-mass spectrometry method for the quantification of the soluble receptor of advanced glycation end-products (sRAGE) in serum using immunopurification in a 96-well plate format

Abstract

The study of proteins is central to unraveling (patho)physiological processes and has contributed greatly to our understanding of biological systems. Corresponding studies often employ procedures to enrich proteins from their biological matrix using antibodies or other affinity binders coupled to beads with a large surface area and a correspondingly high binding capacity. Striving for maximal binding capacity may, however, not always be required or desirable, for example for proteins of low abundance. Here we describe a simplified immunoprecipitation in 96-well ELISA format (IPE) approach for fast and easy enrichment of proteins. The applicability of this approach for enriching low-abundant proteins was demonstrated by an IPE-based quantitative workflow using liquid chromatography-mass spectrometry (LC-MS) for the soluble Receptor of Advanced Glycation End-products (sRAGE), a promising biomarker in chronic obstructive pulmonary disease (COPD). The method was validated according to U.S. Food and Drug Administration (FDA) and European Medicines Agency (EMA) guidelines and enabled accurate quantitation of sRAGE between 0.1 and 10 ng/mL in 50 µL serum. The assay showed substantial correlation with the two most commonly-used sRAGE immunoassays (ELISAs) (R2-values between 0.7 and 0.8). However, the LC-MS method reported 2–4 times higher sRAGE levels compared to the ELISAs, which is largely due to a suboptimal amount of capturing antibody and/or calibration strategy used by the immunoassays. In conclusion, our simplified IPE approach proved to be an efficient strategy for enriching the low-abundant protein sRAGE from serum and may provide an easy to use platform for enriching other (low-abundant) proteins from complex, biological matrices.