Overview
Targeted proteomics focuses on quantifying a predefined set of proteins with high sensitivity, specificity, and throughput, in contrast to discovery proteomics which aims for broad, untargeted coverage. The most widely adopted technique is selected reaction monitoring (SRM, also called multiple reaction monitoring or MRM) performed on triple quadrupole mass spectrometers. SRM assays target specific peptide-precursor-to-fragment transitions, known as proteotypic peptides, that uniquely represent the protein of interest. This approach delivers quantitative precision comparable to immunoassays while offering higher multiplexing capability and faster assay development.
Methods
Assay development begins with the selection of proteotypic peptides — peptides that are unique to the target protein, fly well in the mass spectrometer, and produce intense fragment ions. For each peptide, several transitions (precursor ion m/z to fragment ion m/z) are monitored across the chromatographic elution period. Internal standards, typically stable isotope-labeled versions of the target peptides, are spiked into the sample at known concentrations to enable absolute quantification. Parallel reaction monitoring (PRM) on Orbitrap instruments offers a related approach with high-resolution full-spectrum acquisition. Data-independent acquisition (DIA) methods such as SWATH-MS bridge the gap between discovery and targeted analysis by recording all fragment ions from all precursors in a systematic fashion.
Applications
Targeted proteomics is the method of choice for verifying and validating candidate biomarkers discovered in untargeted studies. It is used extensively in clinical research to quantify panels of proteins linked to specific diseases. The assays complement traditional ELISA measurements, offer an alternative to gel-based approaches, and can be multiplexed to track dozens of proteins in a single run. Integration with HPLC separation and mass spectrometry detection ensures robust quantification, while protein quantification assays provide orthogonal validation of total protein concentration.
