SRM and PRM Analysis
Selected reaction monitoring (SRM) analysis relies on the detection of unique transition signatures consisting of peptide-ion and fragment-ion pairs on a triple quadrupole instrument to quantify peptides and proteins across sample sets. Parallel reaction monitoring (PRM) analysis uses a quadrupole-Orbitrap instrument to perform a full scan of each fragmented peptide target. Each instrument has particular advantages for the targeted quantitative proteomics approach.
The primary advantage of SRM experiments is rapid switching between transitions with a set dwell time. The primary advantages of PRM experiments are the added specificity of the analysis due to high resolution and the generally higher sensitivity of the Orbitrap. As a result, most projects will make use of both SRM for panels of high abundance proteins and PRM for panels of lower abundance proteins.
The advantage of SRM and PRM experiments described above is the high specificity of the analysis, but the experiments measure only the predetermined assay targets. High resolution accurate mass (HRAM) experiments are not biased and can be probed for any target peptide at any time, so the HRAM dataset is a valuable resource with the potential to measure large numbers of additional proteins without the need to repeat the initial biological experiment.
The HRAM dataset also serves as a backup experiment that can enhance targeted SRM and PRM data with important results verified, if needed, by investigating additional peptides.
Targeted Assay Development
The facility currently has approximately 500 assays available for mouse and rat, with additional assays in human, yeast, fruit fly, and worm. We organize assays into panels of approximately 40 assays mostly based on different biochemical pathways.
Commonly used panels include glycolysis, gluconeogenesis, Krebs cycle, mitochondrial and peroxisomal beta oxidation, proteostatsis, etc. In principle, a targeted assay can be created for any protein from any organism with a sequenced genome.