Multiplex Immuno-MALDI-TOF MS for Targeted Quantification of Protein Biomarkers and Their Proteoforms Related to Inflammation and Renal Dysfunction

摘要

Circulating proteins are widely used as biomarkers in clinical applications for the diagnosis, prediction, and treatment of numerous diseases. Immunoassays are the most common technologies for quantification of protein biomarkers and exist in various formats. Traditional immunoassays offer sensitive and fast analyses but cannot differentiate between proteoforms. Protein microheterogeneity, mainly due to post-translational modification, has been recognized as a fingerprint for different pathologies, and knowledge about proteoforms is an important step toward personalized medicine. Mass spectrometry (MS) has emerged to be a powerful technique for the characterization and quantification of proteoforms. We have established a novel four-plex immunoassay based on Matrix-Assisted Laser Desorption/Ionization Time-Of-Flight (MALDI-TOF) MS for the targeted quantification of the inflammatory markers C-reactive protein (CRP), serum amyloid A (SAA), and calprotectin (S100A8/9) and the kidney function marker cystatin C (CysC). Antibodies were covalently bound to superparamagnetic beads, which delivered robust and fast sample processing. Polyhistidine-tagged recombinant target proteins were used as internal standards for quantification. Our method identified a number of proteoforms for SAA ( n = 11), S100A8/9 ( n = 4) and CysC ( n = 4). The assay was characterized by low limits of detection (0.01?0.06 μg/mL) and low coefficients of variation (3.8–9.4%). Method validation demonstrated good between-assay agreement with immuno-turbidimetry ( R ~(2) = 0.963 for CRP), ELISA ( R ~(2) = 0.958 for SAA; R ~(2) = 0.913 for S100A8/9), and nephelometry ( R ~(2) = 0.963 for CysC). The low sample consumption of 20 μL and the high sample throughput of 384 samples per day make this targeted immuno-MALDI approach suited for assessment of inflammatory and renal status in large cohort studies based on precious biobanks samples.