BRIMS Center high-throughput translational proteomics workflow for sorting through long lists of biomarkers to obtain the short list of useful candidates.
The typical translational proteomics experiment requires the following steps:
- Discovery: sample characterization (protein identification) in small sample cohorts
- Data processing, extraction and identification of differentially abundant proteins across sample groups in small to medium sample cohorts
- Biological interpretation and definition of a candidate target list
- Targeted quantification of candidate targets in larger sample cohorts for verification and validation
First, samples are analyzed using LC-MS/MS. Thermo Scientific™ Pinpoint™ software is used to generate a database of differentially expressed proteins – putative biomarkers.
Next Pinpoint software mines the database to generate a list of target proteins for subsequent SRM assays to verify the putative biomarkers. Pinpoint makes it easy to develop and optimize high-throughput, quantitative, multiplexed SRM assays. To optimize the SRM assays, iterative triple quadrupole MS analyses are performed.
Finally, the putative biomarkers are verified by interrogating multiple clinical research samples using the optimized, multiplexed SRM assays.