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Protein biomarker discovery has inherent challenges linked to the validation of the analytical method used or to the impact of biological matrices. Matrix influences must be mastered to guarantee the reliability of the identified biomarkers to monitor human diseases. In this study, multiplexed mass spectrometry assays in selected reaction monitoring (SRM) mode have been developed to measure 107 inflammatory putative proteins in matched serum and plasma from 36 ICU trauma patients. The assays' validation directly in clinical samples was shown to be valuable to manage intersample variability. Using the validation process developed here, assays were validated for 58 biomarkers in serum, 57 in plasma, and 55 in both matrices. Correlation analyses demonstrated that the quantitation using SRM of most of the validated biomarkers (45/55) was impacted by the biological matrix and that the matrix impact was biomarker-dependent. Among the 45 impacted biomarkers, 23 were nevertheless correlated between serum and plasma, whereas the quantitation was shown to be equivalent in both for the 10 last proteins. Matrix selection using SRM is therefore suggested to be suitable prior to clinical evaluation of biomarkers in a large cohort of patients.

Impact of Serum and Plasma Matrices on the Titration of Human Inflammatory Biomarkers Using Analytically Validated SRM Assays