Multiple reaction monitoring (MRM), sometimes referred to as selective reaction monitoring (SRM), is a mass spectrometry method that can target selective peptides for the detection and quantitation of a protein. Compared to traditional ELISA, MRM assays have a number of advantages including ease in multiplexing several proteins in the same assay and independence from the necessity for high-quality, expensive, and at times unreliable antibodies. Furthermore, MRM assays can be developed to quantify multiple proteoforms of a single protein allowing the quantification of allelic expression of a particular sequence polymorphism, protein isoform, as well as determining site occupancy of posttranslational modification(s). In this chapter, we describe our workflow for target peptide selection, assay optimization, and acquisition multiplexing. Our workflow is presented using the example of constrained MRM assays developed for the serum protein ApoL1 in its various proteoforms to highlight the specific technical considerations necessary for the difficult task of quantifying peptide targets based on highly specific amino acid sequences by MRM.
Keywords: APO L1; Mass spectrometry; Multiple reaction monitoring; Quantification; Selective reaction monitoring.