Technical considerations for large-scale parallel reaction monitoring analysis

Sebastien Gallien, Adele Bourmaud, Sang Yoon Kim, Bruno Domon*

*Corresponding author for this work

Research output: Contribution to journalArticleResearchpeer-review

133 Citations (Scopus)


Targeted methods have gained acceptance among proteomics community to perform quantitative experiments. However, the current reference to conduct such experiments relies on selected reaction monitoring (SRM) analyses performed on triple quadrupole mass spectrometers, although it suffers from some limitations. First, the low resolution quadrupole mass analyzers do not present enough selectivity to discriminate the analytes from interferences commonly encountered in biological samples. Second, the number of peptides monitored in one single experiment often remains limited. The introduction of high resolution/accurate mass instruments with fast sequencing capabilities has enabled the development of novel quantitative methods. More specifically, the new quadrupole-orbitrap mass spectrometer operated in parallel reaction monitoring (PRM) mode showed detection and quantification performances similar or better than those obtained in SRM, due to the increased selectivity of the high-resolution orbitrap mass analyzer. The versatility of the instrument, with its ability to multiplex the selection of precursor ions and to operate with varying quadrupole isolation windows, has enabled the design of large-scale experiments, which require the optimization of several acquisition parameters to maintain high performance. It includes the adjustments of the fill time of the trapping device and the tight scheduling of elution times of the peptides, ideally adjusted on-the-fly. Biological significance: The present study constitutes a valuable baseline for the proteomics community to better control the trade-off between sensitivity and number of analyzed peptides in large-scale parallel reaction monitoring (PRM) experiments performed on a quadrupole-orbitrap instrument. A standard acquisition method requires careful setting of the parameters, namely the fill time in accordance with the control of the resolving power and the degree of multiplexing on one hand, and the quadrupole selection window on the other hand. This study helps in establishing acquisition parameters for large-scale PRM experiments, while maintaining sufficient sensitivity. In addition, the real-time correction of the scheduled peptide monitoring windows, to compensate for possible elution time drift, was explored. This approach supports the use of narrowed monitoring windows in PRM analyses, which greatly scale up the number of peptides targeted in a single LC-MS experiment. The broad application of the presented approaches by the community is likely to allow the establishment of an unprecedented scale for targeted proteomics, thus matching the pressing demand of systems biology or biomarker evaluation studies.

Original languageEnglish
Pages (from-to)147-159
Number of pages13
JournalJournal of Proteomics
Publication statusPublished - 4 Apr 2014


  • High resolution/accurate mass
  • Multiple reaction monitoring
  • On-the-fly correction of elution time drifts
  • Parallel reaction monitoring
  • Selected reaction monitoring
  • Targeted proteomics


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