Plasma preparation to measure FDA‐approved protein markers by selected reaction monitoring

Abstract

Abstract BackgroundThe development of commercially available panels for human blood plasma screening via selected reaction monitoring (SRM) offers reliable, cost‐efficient and highly‐standardized discovery and validation of protein biomarkers. However, protein detection by SRM can be hampered by interfering peptide fragment ions. To estimate the influence of interference on protein detection, we performed different types of sample preparation and implemented SRM measurements for well‐characterized protein targets approved by the US Food and Drug Administration. MethodsWe used the PlasmaDeepDive™ SRM assay from BiognoSYS AG for absolute quantification of 18 proteins in 19 samples of human plasma using three different protocols for sample preparation. SRM measurements were performed using iRT standards for retention time normalization and isotopically‐labeled reference peptides for absolute quantification. SpectroDive™ software was used for automated detection of reliable peak groups. ResultsFourteen targeted proteins were quantitatively measured in more than half of the samples. Depletion of highly‐abundant plasma proteins and peptide fraction clean‐up on centrifuge plates resulted in detection of all 18 targeted proteins in femtomolar to picomolar concentrations. ConclusionsIt was shown that commercially designed SRM kits are suitable for SRM detection of well‐established plasma/serum biomarkers.

Keywords

• Proteomics
• Biomarkers
• Mass spectrometry
• Selected reaction monitoring