Stage-One Assay Development
The objective of stage-one assay development is to develop an assay that will be used to confirm candidate biomarkers. Having the necessary data on the candidate proteins captured during biomarker discovery allows for quick assay development. This information may also be provided by a database of proteins previously detected by mass spectrometry. NextGen Sciences has such a database of protein biomarker information, called biomarkerlibrary™.
The assay platform used in this workflow is multiple reaction monitoring (MRM) for peptides (Figure 3). This is a mass spectrometry-based assay that targets specific surrogate peptides in a sample. The approach brings high specificity for each protein in the assay. MRM assays do not require antibodies and can be designed to monitor a specific protein, single or multiple isoforms of a protein, and even a single amino acid mutation within the protein(s) being measured.
Peptides for each protein are selected based on their known amino acid composition, response factor, hydrophobicity, chromatographic peak shape, and product ion distribution. A set of parent ion/product ion combinations (termed transitions) per peptide is tested; typically many more peptides and transitions are chosen than used in the final assay.
During assay development, positive correlation is required between the transition ratios obtained during MRM analysis and with the data from the discovery experiment or the data in the biomarker library. This ensures that a specific signal is unique to the biomarker protein. The peptide and transition list is refined at this point.
Next, analytical reproducibility of each peptide is assessed, by analyzing one preparation of the same sample many times in the mass spectrometer. Technical reproducibility of each peptide is then assessed by measuring the peptide performance from multiple preparations of the same sample. The peptide and transition list may again be refined at this point. Once all peptides have been selected they can be monitored in the assay.
A key feature of the MRM platform is that many peptides and their transitions can be multiplexed in a single chromatographic run. The ability to quickly develop a multiplexed assay for many proteins allows for facile initial biomarker testing. The timeline for this stage is typically four to six weeks.