Using pure protein to build a multiple reaction monitoring mass spectrometry assay for targeted detection and quantitation.

Multiple reaction monitoring (MRM) is an increasingly popular mass spectrometry-based method to simultaneously detect and quantify multiple proteins. MRM is particularly useful for validating biomarkers discovered with a mass spectrometer and any analite discovered by MS can be monitored by MR because an MRM assay can be developed without the need to generate specific antibodies. In this chapter, we present a robust and systematic procedure to rapidly build a high-sensitivity MRM assay using purified protein as the starting material. Theoretical digestion of the protein with trypsin is used to identify mass spectrometry--compatible peptides and to generate preliminary MRM transitions to detect these peptides. Peptides generated by trypsin cleavage of the actual protein are then run on a liquid chromatography column coupled to a triple quadrupole mass spectrometer, which is programmed with the preliminary transitions. Whenever a transition is detected, it triggers dissociation of the corresponding peptide and collection of a full mass range scan of the resulting fragment ions. From this scan, fragment ions yielding the strongest and most reproducible signals are utilized to design empirical MRM transitions. The assay is further refined by optimizing the collision energy and creating a standard curve to measure sensitivity. Once MRM transitions have been established for a particular protein, they can be combined with transitions for other target proteins to create multiplex assays and used to quantify proteins in samples arising from serum, urine, subcellular fractions, or any other specemen of interest.