Unlike the unbiased discovery approach, which attempts to detect all proteins in a biological sample, MRM is highly selective (targeted), allowing researchers to look for specific peptides or protein fragments of interest. Use of MRM, which was selected as Method of the Year by Nature Methods in 2012, has been increasing dramatically due to its high sensitivity and specificity and its ability to provide absolute quantitation. Because there is no need for expensive and time-consuming antibody development, this approach also offers significant time and cost savings over antibody-based methods.
Caprion uses a fit-for-purpose approach for developing MRM-based assays. Assays range from research-grade assays that measure 100s of proteins for rapid discovery or verification of biomarker candidates to assays that provide absolute quantitation of a single or a few proteins for diagnostics or clinical applications, performed under GLP.
Caprion’s MRM-based offerings include:
- Off-the shelf multiplexed panels for CNS, immune response, toxicology, and plasma/serum proteins
- Rapid development of custom panels
- Fit-for-purpose assays for non-clinical and clinical use
- Validated assays for clinical use (biomarker quantitation or PK)
The MRM assay development process is described below.
Protein targets can be selected from multiple sources, including prior “omics” studies, literature, and pathway analysis (1). Following in silico analysis to predict the best peptides for MRM analysis (2), peptides are synthesized (3) and are typically isotope labeled to facilitate accurate quantitation over multiple sample batches.
Synthetic peptides are then analyzed to determine the best transitions (or fragment ions) to monitor (4). Further optimization is performed to assess sensitivity and dynamic range (5) before characterizing assay performance in the relevant sample matrix (6). Controls and standards are included as needed to provide fit-for-purpose assays spanning research through clinical use.
How MRM Mass Spectrometry Works
MRM uses triple quadrupole mass spectrometers coupled to liquid chromatography to enable greater specificity, sensitivity, and quantitation of analytes of interest. In the first quadrupole (Q1), a specific peptide that corresponds to a protein of interest is selected. The peptide is then fragmented in the second quadrupole (Q2) and a filter is applied to allow a specific fragment, also referred to as a transition, to enter into the third quadrupole (Q3) where its intensity is measured. Because of the double selection approach, which improves signal-to-noise and reduces interference, and the multiple measurements per protein, MRM is one of the most specific assays for protein measurement.