Stage-Two Assay Development
While determination of the relative protein levels is sufficient for biomarker confirmation, protein concentration levels are required for biomarker validation. In order to achieve this, the relative quantitation assay from stage one is further developed to provide concentration values for each protein—often called absolute quantitation.
Isotopically labeled peptide standards (typically 13C or 15N labeled amino acids), that behave identically to the native peptide except for a shift in mass, are used to convert mass spectrometry data (analyte peak area) to protein concentrations. Separate calibration curves for each peptide included in the assay are established, each including six calibrators that cover the expected concentration range in the samples. The curves are run under the exact conditions established during assay development.
The MRM assay is validated on a fit-for-purpose basis; since small molecule MRM has been used for over 20 years for clinical samples, detailed and specific validation steps have been established that can be applied to MRM for peptides. The timeline for this stage is typically two to six months.
Stage-Two Biomarker Testing
The objective of stage-two testing is to validate the biomarkers. Data are reported as the concentration of each protein in each sample. This step of biomarker development involves hundreds of samples tested in batches. The assay is robust enough for the data to be highly reproducible over time.
NextGen Sciences is currently using the workflow described in this tutorial to develop protein biomarker assays for pharmaceutical and biotechnology companies. This has helped relieve the bottleneck of assay development. We have demonstrated that the MRM platform can be used to develop multiplex assays in the timelines presented in this workflow.