Method Validation by CPTAC Guidelines for Multi-protein Marker Assays Using Multiple Reaction Monitoring-mass Spectrometry

Quantifying multiple protein biomarkers in a blood sample at one time has many advantages for diagnosing human diseases. In this study, 34 multiplex assays by multiple reaction monitoring-mass spectrometry (MRM-MS) for serum biomarkers were characterized according to Clinical Proteomic Tumor Analysis Consortium (CPTAC) guidelines. The assays revealed that the median lower limit of quantitation (LLOQ) was 0.37 fmol/μL (16.0 ng/mL) and that the median total coefficient of variation (CV) was 18.2%, 12.2%, and 10.6% in the low-,medium-, and high-quality control (QC) samples. With regard to selectivity, the median mean differences in slope and concentration were 2.1% and 4.3%, respectively. The median values for all CVs and %difference from the nominal concentration for stability were 9.5% and 2.7% in low-QC and 3.8% and 3.1% in medium-QC. The median total CV was 9.8% in the reproducibility. Finally, 17 protein-based biomarker assays were reliable and transferrable for preclinical purposes per CPTAC guidelines.