Multiple Reaction Monitoring (MRM) // Multiple Reaction Monitoring (MRM) for Protein and Biomarker Discovery, Validation and Quantification
Multiple reaction monitoring (MRM) using mass spectrometry is a highly sensitive and selective method for the targeted quantitation of protein/peptide abundances in complex biological samples. MRM mass spec has commonly been used for the analysis of small molecules.
Unlike traditional mass spectrometry, which attempts to detect all proteins in a biological sample in an unfocused fashion, Multiple Reaction Monitoring (MRM) is highly selective (targeted), allowing researchers to fine tune an instrument to specifically look for peptides, or protein fragments, of interest. This approach allows for greater specificity, sensitivity, speed and quantitation of an analyte of interest (eg. biomarker candidate). The MRM platform technology allows for targeted analysis of proteins of interest rather than sifting through enormous amounts of data that typically result from non-targeted studies.
Our MS-based approach provides absolute structural specificity for the analyte (protein), and in combination with appropriate stable isotope-labeled internal standards (SIS), it provides absolute quantitation of protein concentration. MRM analysis is capable of sensitive (attomole level) and absolute determination of peptide concentrations across a wide concentration scale spanning a dynamic range covering 3 to 4 orders of magnitude. Additionally, MRM-based quantitation with SIS peptides does not “miss” peptides because the SIS peptide must be detected in every sample: this means that if an endogenous peptide is not observed then it is below the limit of detection.
Our PeptiQuant™ MRM assays are based on the selection of specific tryptic peptides as stoichiometric representatives of the proteins from which they are cleaved. The tryptic peptides are quantitated against a spiked internal standard (a synthetic stable isotope-labeled peptide) to yield a measure of protein concentration). This enables the performance of each assay to be assessed for limits of detection/quantitation and linearity. Therefore our MRM assays require only a knowledge of the protein sequences, the masses of the selected peptides and their fragment ions.
Mass spectrometry provides an alternative to ELISA and other antibody based assay, by relying on the discriminating power of mass analyzers to select a specific analyte and on ion current measurements to determine quantitation.
MRM Proteomics Platform technology allows researchers to select peptides of interest while all other peptides are filtered out. Peptides are detected by mass spectrometry analysis and the exact concentration can be determined (see below).
Scheme of a Multiple Reaction Monitoring (MRM) experiment. The first mass analyzer (Q1) is set to only transmit the parent weight of a protein, the collision energy is optimized to produce a diagnostic charged fragment of a protein fragment (peptide) in the second mass analyzer (Q2), and the third mass analyzer (Q3) is set to transmit this diagnostic peptide fragment only. Therefore, only this exact peptide transition is detected.