Targeted proteomics using multiple reaction monitoring (MRM) mass spectrometry is becoming an increasingly popular choice for quantitating specific sets of proteins in biological systems. While MRM represents the most sensitive MS approach to pathway mapping, challenges arise due to the need to construct MRM assays that are unique for the peptide of interest. Furthermore, the assay development steps can be slow as due to the need translate across different instrument platforms (i.e. Q-Tof to QQQ).
Recent innovations in the TripleTOF 5600 System have enabled a new acquisition strategy, where MS/MS spectra are collected at high resolution in a TOF analyzer, then fragment ions are extracted post-acquisition to generate high resolution MRM-like data. The technique, termed MRM-HR quantification, is sensitive and fast enough to enable quantitative performance similar to high end triple quadrupole instruments, without the hassles of assay translation across instrument platforms.
Example of a TOF MS spectrum where a targeted analyte previously seen in Information Dependant Aquisition (IDA) run is selected for high-intensity, high resolution fragment ions unique to the targeted peptide. Typically MS spectra are acquired using 250 ms accumulation time per spectrum.
Extracted ion chromatogram (XIC) of the targeted peptide generated post-acquisition. High resolution extraction of fragment ions provides higher specificity quantitation in complex matrices