Workflows » High resolution MRM
MRM (multiple reaction monitoring) also called SRM (selected reaction monitoring) is a targeted data acquisition where known precursor ion signatures such as precursor m/z, chromatographic retention time and specific CID-based fragment ion m/z values are used for compound detection and quantification.
MRMs are usually performed on triple quadrupole (QqQ) instruments where the first quadrupole serves as a mass selective filter open for only the selected m/z value (often within one unit resolution), the second as a collision quadrupole to generate CID-based fragment ions of the selected precursor m/z and the third as a second mass filter to only select one fragment ion at a time. In the context of MRM fragment ions specific for the compound of interest are called transitions. To gain greater confidence on detection and quantification usually two or more transitions are selected per precursor ion. The aforementioned procedure has to be repeated for every transition in a QqQ instrument. Modern instruments have the required speed to cycle through many precursor ions with multiple transitions within a short time window to keep up with the sharp chromatography of contemporary uHPLC-systems.
Alternatively to the classical MRM performed on QqQ instruments hybrid QqTOF-type instruments such as the TripleTOF 5600+ can acquire high resolution MRMs. In a high resolution MRM assay the first two quadrupoles basically operate equally to those in a QqQ instrument followed by monitoring all transitions of a selected precursor in a single high resolution fragment ion spectrum recorded by the TOF analyser. High resolution MRM assays provide higher confidence detection because all possible transitions are monitored. Consequently in targeted proteomics fragment ion spectra from high resolution MRM acquisitions can also be used for protein identification through software-based spectrum interpretation using database-dependent search engines. Such a hypothesis-driven approach can be used to try to verify the presence of proteins that failed to be detected by untargeted discovery approaches. Using software-tools such as Skyline a list of suitable tryptic peptides from a number of target proteins and their most likely transitions can be predicted and fed into the analysis software of the mass spectrometer for high resolution MRM analysis. Using the TripleTOF 5600 + instrument we are able to screen for roughly 20-30 peptides in an unscheduled MRM approach. If retention times are known many more precursors can be selected in a scheduled high resolution MRM experiment. In such a scheduled MRM assay precursor are only selected during their specific retention time windows to keep the cycle time of the mass spectrometer as low as possible.