Targeted quantification of C-reactive protein and cystatin c and its variants by immuno-MALDI-MS

The most common technologies for quantitative determination of protein biomarkers are immunoassays, which exist in various formats. Immunoassays offer sensitive and fast protein quantification, but can hardly discriminate between protein variants. Post-translational modifications and genetic variants increase protein microheterogeneity and may play important roles in biological processes. Mass spectrometry combined with immunoaffinity enrichment detects protein microheterogeneity and can quantify different isoforms. We here present an immuno-MALDI-MS approach for the combined quantification of two important biomarkers of inflammation and renal function, C-reactive protein (CRP) and cystatin C, respectively. Antibodies were immobilized onto reversed-phase tips, which allows easy and flexible sample processing. Quantification was performed in singleplex and duplex assays, and characteristics were evaluated for different internal standards, i.e., PEGylated and polyhistidine-tagged proteins. The best performances were obtained for polyhistidine-tagged standards with respect to limits of detection (CRP, 0.10 μg/mL; cystatin C, 0.003 μg/mL) and coefficients of variation (CRP, 2.4-7.0%; cystatin C, 3.0-8.9%). The methods were benchmarked against immunoturbidimetry and nephelometry and demonstrated good between-assay agreement (R(2) = 0.989 for CRP; R(2) = 0.939 for cystatin C). Several variants of cystatin C were identified and quantified, while none were observed for CRP. This immuno-MALDI method describes a novel approach for targeted quantitative investigation of protein microheterogeneity and is well suited for assessment of biomarker status in precious samples from biobanks due to its low sample consumption.