![]() |
![]() |
Selected/Multiple reaction monitoring assays, conducted on triple quadrupole instruments, can be coupled to liquid chromatography for the analysis of complex proteome digests. In SRM/MRM assays the first (Q1) and last (Q3) mass analyzers of a triple quadrupole mass spectrometer are used as mass filters to isolate a peptide ion and a corresponding fragment ion. The signal of the fragment ion is then monitored over the chromatographic elution time (Fig. 1). The selectivity resulting from the two filtering stages, combined with the high duty cycle, results in quantitative analyses with unmatched sensitivity and specificity. The specific pairs of m/z values associated to the precursor and fragment ions selected are referred to as "transitions" and effectively constitute mass spectrometric assays that allow to identify and quantify a specific peptide and, by inference, the corresponding protein in a complex protein digest. ![]() Figure 1. The process of establishing a SRM/MRM assay for a protein consists of a number of steps: Overall, this is a lengthy and iterative process, but, once an MRM assay for a protein is established, it becomes universally useful, i.e. the tedious assay development process needs to be performed only once, for a given type of mass spectrometer and fragmentation mechanism (e.g. collision induced-dissociation). In the MRMAtlas each protein assay is presented as a set of optimal MRM coordinates for the peptide(s) that represent a protein. Peptide identifications have been validated by acquiring the corresponding tandem mass spectra on the triple quadrupole mass spectrometer, which can be viewed as single or consensus spectra. The final assay coordinates can be directly downloaded in Excel table-format which can be directly pasted into a MRM/SRM method of a triple quadrupole instrument and used to specifically detect and quantify the protein of interest in a complex protein digest. (Adapted from: A. Schimdt, P. Picotti and R. Aebersold Proteomeanalyse und systembiologie BIOspektrum, 1/2008, S. 44) |
联系客服