Parallel Reaction Monitoring (PRM) is a targeted proteomics method used to measure specific proteins within a complex mixture like blood or tissue. As a hypothesis-driven approach, scientists pre-select a list of proteins to analyze. The primary goal of PRM is to provide highly confident and accurate quantification of these targets. This technique combines high selectivity and sensitivity, enabling the reliable measurement of proteins, even those present in low amounts.
The PRM Workflow
The PRM workflow is a multi-step process that begins with planning and sample preparation. Scientists select specific peptides, which are unique segments of the proteins they intend to study, to serve as surrogates. This selection is guided by previous experiments or public databases, and the chosen peptides should be easy to detect and free of frequently modified amino acids.
The prepared sample is introduced into a mass spectrometer. Inside, a component called a quadrupole isolates the specific peptide ion of interest, known as the precursor ion. This isolation step acts like a filter, removing thousands of other molecules in the sample to focus only on the targeted peptide.
Following isolation, the precursor ion is fragmented into smaller pieces using higher-energy collisional dissociation (HCD). The resulting fragment ions are then detected simultaneously in a high-resolution mass analyzer, like an Orbitrap. This parallel detection of all fragments provides a detailed and specific “fingerprint” of the original peptide, ensuring high confidence in its identification.
PRM vs. SRM
The primary distinction between PRM and its predecessor, Selected Reaction Monitoring (SRM), is how they detect fragment ions. SRM isolates a precursor ion and fragments it, but then uses a second mass filter to monitor only a few pre-selected fragment ions, called transitions. This makes SRM sensitive, but it is “blind” to other fragments, which can lead to inaccurate measurements if an interfering compound is present.
PRM, in contrast, uses a high-resolution mass analyzer to capture the entire spectrum of fragment ions at once. This comprehensive detection provides a richer dataset for more confident identification of the target peptide. By observing the full “fingerprint,” scientists can distinguish the true signal from background noise or interfering molecules. This view also allows researchers to decide which fragment ions to use for quantification after data collection.
SRM is often favored for its raw sensitivity and high throughput in established, routine assays. PRM is the method of choice when the goals are high specificity and flexibility, especially when analyzing complex samples or developing new methods. The investment in developing an SRM method is also greater, as it requires upfront validation of which transitions to monitor.
Interpreting PRM Data
Processing PRM data begins with identification. This step involves matching the acquired fragment ion spectrum to a reference spectrum from a library or theoretical model. A high degree of similarity between the measured and reference spectra provides strong evidence that the correct peptide was detected.
Retention time, the specific time a peptide takes to travel through the liquid chromatography system, adds another layer of confidence. This time is highly reproducible for a given peptide under consistent conditions. The high mass accuracy of PRM instruments also allows for the use of very narrow mass windows when extracting data, which minimizes interference.
Quantification is achieved by measuring the signal intensity of fragment ions, which creates a chromatographic peak. The area under this peak is directly proportional to the amount of the peptide in the sample. Software tools like Skyline are used to integrate these peak areas and compare peptide abundance across samples.
Applications in Scientific Research
One common use for PRM is validating potential disease biomarkers. If a discovery study identifies proteins at different levels in diseased versus healthy individuals, PRM can measure these proteins in more patient samples to confirm their link to the disease.
The technique is well-suited for studying post-translational modifications (PTMs), which are chemical changes that can turn protein functions on or off. PRM can quantify the modified form of a peptide relative to its unmodified version, providing insights into cellular signaling. This is useful in fields like cancer research, where signaling pathways are often dysregulated.
PRM is applied in systems biology and drug development to understand how biological systems respond to treatments. Researchers can measure changes in protein levels within a cellular pathway after introducing a drug, helping to confirm its mechanism of action. Its precision also makes it valuable for quantifying proteins in biological fluids like plasma or urine for clinical research.