Multiple Reaction Monitoring (MRM)
Multiple reaction monitoring (MRM) coupled with stable isotope dilution mass spectrometry (SID-MS) is a highly specific and sensitive mass spectrometry technique for quantitative measurement of target proteins. This targeted MS analysis 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. This technique requires a triple quadrupole mass spectrometer.
At Dundee Proteomics Facility we use a Qtrap 4000 (AB Sciex), or TSQ Quantiva (Thermo Scientific) where the precursor peptide mass is selected in Q1 and diagnostic CID fragment ions are selected in Q3. A signal is registered only when a pre-defined fragment ion arises from the pre-defined precursor. For absolute quantitation, and maximum sensitivity synthetic, isotopically labelled peptide standards are needed to optimize MS parameters and also to build a standard curve. This quantification method could be used for biomarker validation, measurement of phosphorylation stoichiometry ….etc.
For quantification of proteins at least two ionisable unique peptides could be chosen from the tryptic digest of the protein of interest. Also, two to three transitions per peptide are necessary for the MRM method. For complex samples and low abundant proteins (phospho-proteins) the workflow may involve an enrichment of the protein of interest using antibodies, the resulting pull down could be either run on gel electrophoresis to cut the band of interest (in-gel digestion) or used as it is for in-solution digestion. Below is a typical workflow for protein quantification.