Determination of the concentration range for 267 proteins from 21 lots of commercial human plasma using highly multiplexed multiple reaction monitoring mass spectrometry

Multiple reaction monitoring (MRM) is a key tool for biomarker validation and the translation of potential biomarkers into the clinic. To demonstrate the applicability of MRM towards achieving this goal, we set out to determine the concentration ranges of 267 plasma proteins, including 61 FDA-approved/LDT developed biomarkers, in 21 commercial human plasma lots, as well as to assess accuracy and precision. Each target protein was quantified by calculating the area ratio of the endogenous tryptic target peptide to its stable isotope-labelled internal standard equivalent and compared to a standard curve. This highly multiplexed approach utilized a standard-flow UHPLC system linked to a triple quadrupole. All samples were analyzed across three separate days and assessed for robustness and accuracy. The standard curves and quality control samples showed excellent performance, with >93% of standards and QCs meeting the acceptance criteria. A total of 248 proteins were able to be quantified in at least one sample on at least one of the three days, with 111 proteins being quantified in all 21 samples on all three days. The protein concentrations across all proteins covered six orders of magnitude. Furthermore, excellent three-day precision was demonstrated with 86% of CVs falling below 15%. Overall, the protein concentration differences ranged from 1.1-fold for metalloproteinase inhibitor 2, to 69-fold for serum amyloid A-1/A-2.