MRM (Multiple Reaction Monitoring)
Biomarker discovery produces lengthy lists of candidate proteins that are detected differentially in cases versus controls and require further verification in large numbers of patient samples. Verification of candidate proteins requires targeted, multiplexed assays to screen and quantify proteins in patient plasma samples with high sensitivity, absolute specificity and sufficient throughput.
Multiple reaction monitoring (MRM) coupled with stable isotope dilution mass spectrometry (SID-MS) using a triple quadrupole mass spectrometer is a powerful method for quantitative measurement of target proteins. It has been a principal tool for quantification of small molecules in clinical chemistry for number of decades. MS-based quantitative assays have the necessary characteristics required for verification studies, namely: high specificity, sensitivity, multiplexing capability, and precision.
In MRM mode, two stages of mass filtering are employed on a triple quadrupole mass spectrometer. In the first stage, an ion of interest (the precursor) is preselected in Q1 and induced to fragment by collisional excitation with a neutral gas in a pressurized collision cell (Q2). In the second stage, instead of obtaining full scan ms/ms where all the possible fragment ions derived from the precursor are mass analyzed in Q3, only a small number of sequence-specific fragment ions (transition ions) are mass analyzed in Q3. This targeted MS analysis using MRM enhances the lower detection limit for peptides by up to 100 fold (as compared to full scan ms/ms analysis) by allowing rapid and continuous monitoring of the specific ions of interest.
MRM methods, in principal, provide both absolute structural specificity for the analyte and relative or absolute measurement of analyte concentration when stable, isotopically-labeled standards are added to a sample in known quantities. When a synthetic, stable isotope labeled peptide is used as an internal standard, the concentration can be measured by comparing the signals from the exogenous labeled and endogenous unlabeled species. This can be done because they have the same physicochemical properties and differ only by mass.
MRM assay configuration
MRM assay configuration starts with the selection of “signature” peptides that act as surrogates for the quantification of protein of interest. Scientist in our group apply their knowledge and select typically 2 to 3 peptides per protein for MRM assays. These signature peptides are synthesized with heavy labeled Arginine or Lysine at the C-terminus of the peptide to serve as internal standards in the MRM assay.
The next step in MRM assay configuration is optimization of MS parameters for maximum transmission and sensitivity of each MRM transition. Final MRM assay is set for each peptide to achieve the most sensitivity and avoid interferences present in complex backgrounds.
In order to define lower limits of detection and quantification (LOD and LOQ) calibration curve is generated for each peptide in buffer and a background relevant to the particular study. For a typical calibration curve concentration of unlabeled peptide ranges from subfemtomol to 100s of fmol levels, whereas concentration of heavy labeled internal standard is kept constant.
SID/MRM-MS assays using retention time scheduling (sMRM) allows simultaneous detection and quantification of tens to hundreds of candidate proteins in plasma.
Application of SID/MRM-MS to Biomarker Verification
Many biomarkers of clinical importance reside in picogram/mL to low nanogram/mL range in plasma. If no plasma processing is employed before SID/MRM-MS it is possible to detect and quantify proteins present in plasma at microgram/mL levels due to high complexity and dynamic range of plasma. However, abundant protein depletion followed by limited fractionation at the peptide level prior to SID/MRM-MS achieves LODs and LOQs in low nanogram/mL levels with process CVs ranging from 5% to 20%. Similar performance characteristics are achieved with anti-peptide antibody enrichment (SISCAPA) prior to MRM-MS. This approach greatly simplifies the process since it doesn’t require depletion or fractionation of plasma, however production and qualification of anti-peptide antibodies is more expensive and takes longer. For small number of highly credentialed candidates SISCAPA will be the method of choice as it allows screening large number of patient samples in less time compared with depletion and fractionation approach.
Keshishian, H., T. Addona, et al. (2007) "Quantitative, multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution." Mol Cell Proteomics 6:2212-29. Abstract
Addona, T. A., S. E. Abbatiello, et al. (2009) "Multi-site assessment of the precision and reproducibility of multiple reaction monitoring-based measurements of proteins in plasma." Nat Biotechnol 27:633-41. Abstract