Antibody-free LC-HRMS/MS method for simultaneous quantification of NGAL, CRP and SAA in serum from sepsis patients

Liquid chromatography coupled with high-resolution mass spectrometry (LC-HRMS/MS) is a valuable alternative to ligand-binding assay, enabling specific and accurate quantification of protein biomarkers. We developed a robust antibody-free LC-HRMS/MS method for the multiplex quantification of three sepsis biomarkers in serum: NGAL, CRP and SAA. The method was thoroughly optimized from sample preparation to LC-HRMS/MS analysis, alongside the calibration. Specifically, a modified trichloroacetic acid/isopropanol protein precipitation procedure combined with an optimized Parallel Reaction Monitoring acquisition allowed the quantification of the low abundant NGAL at ng/mL levels. While reference material and reference measurement procedure were available for CRP, no such standards existed for NGAL and SAA. Well-characterized peptide calibrators traceable to the international system of units were developed for NGAL and SAA. The method demonstrated suitable trueness and precision for the quantification of NGAL, CRP, and SAA with coefficients of variation (CV%) ranging from 1.6 % to 22.4 % and bias between -12.6 % and +18.0 %. Successful application to pooled serum samples illustrated the method's effectiveness. Our results pave the way toward the development of reference systems for additional sepsis biomarkers.