Multiple reaction monitoring (MRM) is a highly sensitive method of targeted mass spectrometry (MS) that can be used to selectively detect and quantify peptides based on the screening of specified precursor peptide-to-fragment ion transitions. MRM-MS sensitivity depends critically on the tuning of instrument parameters, such as collision energy and cone voltage, for the generation of maximal product ion signal. Although generalized equations and values exist for such instrument parameters, there is no clear indication that optimal signal can be reliably produced for all types of MRM transitions using such an algorithmic approach. To address this issue, we have devised a workflow functional on both Waters Quattro Premier and ABI 4000 QTRAP triple quadrupole instruments that allows rapid determination of the optimal value of any programmable instrument parameter for each MRM transition. Here, we demonstrate the strategy for the optimizations of collision energy and cone voltage, but the method could be applied to other instrument parameters, such as declustering potential, as well. The workflow makes use of the incremental adjustment of the precursor and product m/z values at the hundredth decimal place to create a series of MRM targets at different collision energies that can be cycled through in rapid succession within a single run, avoiding any run-to-run variability in execution or comparison. Results are easily visualized and quantified using the MRM software package Mr. M to determine the optimal instrument parameters for each transition.
Multiple reaction monitoring mass spectrometry (MRM-MS) is a technique for high-sensitivity targeted analysis. In proteomics, MRM-MS can be used to monitor and quantify a peptide based on the production of expected fragment peaks from the selected peptide precursor ion. The choice of which fragment ions to monitor in order to achieve maximum sensitivity in MRM-MS can potentially be guided by existing MS/MS spectra. However, because the majority of discovery experiments are performed on ion trap platforms, there is concern in the field regarding the generalizability of these spectra to MRM-MS on a triple quadrupole instrument. In light of this concern, many operators perform an optimization step to determine the most intense fragments for a target peptide on a triple quadrupole mass spectrometer. We have addressed this issue by targeting, on a triple quadrupole, the top six y-ion peaks from ion trap-derived consensus library spectra for 258 doubly charged peptides from three different sample sets and quantifying the observed elution curves. This analysis revealed a strong correlation between the y-ion peak rank order and relative intensity across platforms. This suggests that y-type ions obtained from ion trap-based library spectra are well-suited for generating MRM-MS assays for triple quadrupoles and that optimization is not required for each target peptide. Table 1, a description of the contents of the ISB standard 18 protein mix; Supplemental Table 2, the tabulated AUC and peak intensity data for the full combined data set; Supplemental Figure 1, the results for the rank order permutation test for the three individual data sets; Supplemental Figure 2, the results from the relative intensity randomization test for the three individual data sets; Supplemental Figure 3, the results from an alternative relative intensity randomization test in which the triple quadrupole spectra were randomized by permuting the existing relative intensities for each spectrum; Supplemental Figure 4, the results from a second alternative relative intensity randomization test in which the triple quadrupole spectra were randomized by replacing each peak intensity with a random value from the data set; and Supplemental Analysis, the secondary rank order analysis performed on the 142 excluded peptides. This material is available free of charge via the Internet at
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