Reproducible targeted peptide profiling using highly multiplexed MRM assays

Using SCIEX Triple Quad 7500 system

Christie L Hunter


Using the Scheduled MRM algorithm to build highly multiplexed peptide MRM methods enables a higher number of MRMs to be monitored concurrently while maintaining optimal dwell and cycle times. SCIEX Triple Quad 7500 system performance for large scale targeted peptide quantification was evaluated using microflow chromatography, testing methods up to 4000 MRMs in a single method.



The utility of Multiple Reaction Monitoring (MRM) for targeted protein quantification and biomarker verification/validation studies on triple quadrupole-based MS systems is driven by the renowned sensitivity and selectivity attributes the technique delivers. As more extensive protein panels need to be monitored in a targeted way across multiple samples, higher multiplexing of MRM transitions is becoming essential for throughput. This growing need to run higher numbers of samples in biomarker research projects is also driving a shift from longer nanoflow gradients to faster microflow chromatography runs.

But quantitative robustness remains key, to ensure both large and small biological changes are accurately measured across large sample cohorts. Biological matrices also have a very wide range of protein abundance, which requires the LC-MS/MS system to have high sensitivity as well as wide linear dynamic range.

Here a simulated study was performed to test the performance of the SCIEX Triple Quad 7500 system for large scale targeted peptide quantification. Microflow chromatography was used to explore both longer and short gradients. Methods with up to 4000 MRM transitions were run and the quantitative reproducibility was evaluated.


Key Features of the SCIEX 7500 system for highly multiplexed peptide MRM assays

  • SCIEX Triple Quad 7500 system provides high sensitivity for peptide quantification, with improvements in ion generation due to the OptiFlow Pro ion source with E Lens probe and in ion sampling due to the D Jet ion guide1 
  • OptiFlow Pro ion source provides flexibility and ease of use, with interchangeable probes and electrodes that require minimal user optimization 
  •  Switch to microflow chromatography in minutes 
  • SCIEX OS software 2.0 automates MRM time scheduling with the intelligent Scheduled MRM algorithm, allowing many more analytes to be analyzed in a single run
  • The Scheduled MRM algorithm uses peptide retention times to automatically compute an optimized acquisition method based on a few key parameters provided by the user