Mass spectrometric immunoassay for quantitative determination of protein biomarker isoforms

Protein biomarkers are essential in assessing pathogenic processes. The impetus for finding new biomarkers has been accelerated by the arrival of the "omics" technologies. However, equally important is the rediscovery of existing biomarkers with these new approaches as novel variants can be discovered that can improve their utility. Presented here is a mass spectrometric immunoassay method for quantitative determination of β-2-microglobulin, an established biomarker used in the diagnosis of active rheumatoid arthritis and kidney disease, and its structural variant, cleaved at and deficient in lysine-58 (ΔK58-b2m). β-Lactoglobulin was incorporated into the assay as an internal reference standard, serving as normalization point for β-2-microglobulin quantification. The precision, linearity, and recovery characteristics of the assay were established. The new assay was also benchmarked against existing β-2-microglobulin ELISA. The assay was utilized to determine the individual concentration of β-2-microglobulin and its variant across a larger cohort of samples, demonstrating the ability to simultaneously quantify both proteins.