Christie Hunter


What is the difference between MRM3 vs MS/MS/MS (MS3)?

The MRM3 workflow and the MS3 scan are functionally the same QTRAP system scan, but used with different goals in mind.  The main difference is how these scans are used in the whole MS workflow. With MS3 scans, you can use these in a data dependent mode for discovery where you can trigger MS/MS/MS scans off of observed fragments of precursors from a preceding MS/MS scan. When we perform the MRM3 workflow, we are focusing on quantification. Here we set up the scan to target specific analytes and build an optimized MS/MS/MS scan, which is then performed in a looped fashion across the LC run to generate quantitative data. More detail on the MRM3 scan can be found in this technical note.

The MRM3 data is collected as full scan spectra, so post acquisition we can then perform extracted ion chromatograms (XICs) on the MS/MS/MS fragments. The advantage here is specificity, if there is high background in your MRM experiment, switching to MRM3 and requiring the fragment of a fragment often provides that key specificity increase.

When doing MRM3, you can loop in other scan modes in the same workflow. So if you wanted to perform an MRM experiment targeting a set of analytes, then loop in a few MRM3 experiments for the analytes that require higher selectivity, that is easy to do in SCIEX OS or Analyst software.  And all the data can be processed together using Analytics in SCIEX OS software.

To learn about developing MRM3 assays with SCIEX OS software, refer to this post.

In Analyst software: There is an MRM3 optimization script in Analyst software (under the Scripts menu) that works really well if you have your analyte to infuse. You just infuse the analyte, provide a bit of upfront information and the script will run through all the tuning steps to optimize the MRM3 scan!  You can find info on the script in the user guide, found at C:\Program Files (x86)\Analyst\Help\Software Guides folder on 64-bit computer, or C:\Program Files \Analyst\Help\Software Guides on 32-bit computer.

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