Fig. 1
The immuno-MRM workflow allows for highly specific and sensitive multiplexed quantification of proteins. The assay workflow begins by generating a protein lysate from the biospecimen. Synthetic, cleavable stable isotope-labeled peptides for each targeted sequence are spiked into the sample at a known concentration to act as internal standards. The protein mixture is enzymatically (Lys-C and trypsin) digested to convert the lysate to a mixture of peptides. Custom monoclonal antibodies coupled to magnetic beads are used to enrich the endogenous peptides and labeled standards, and the eluate is analyzed by multiple reaction monitoring-mass spectrometry, targeting multiple transitions (i.e., combinations of precursor/fragment ion pairs). The endogenous analyte peptides and internal standards coelute (i.e., they feature equivalent retention times) and the relative areas of monitored transitions are used to confirm specificity. The amount of endogenous peptide is quantified relative to the heavy standard.
