After you defined the LC part of the instrument method, configure the MS detection.

Prerequisites

  1. The MS instrument is installed and configured in Chromeleon CDS software using a valid license.
  2. In the part of the instrument method for the MS instrument, the Instrument Method Wizard displays the Method Editor interface of the Orbitrap Exploris.

Procedure

  1. In the Method Editor, set the Application Mode to Peptide.
  2. Set the Method Duration to match the run time that you specified in the LC part of the instrument method.
  3. In the Settings area > Infusion Mode, select Liquid Chromatography and enable Advanced Peak Determination.
  4. This provides better determination of peptide charge states and monoisotopic m/z values of isotopic envelopes. Keep the Default Charge State equal to 2.
  5. Select one of the options for Internal Mass Calibration. Refer to the Orbitrap Exploris Series Tune Help for details of the choices that you can make.
  6. In the Global Parameters tab, select Ion Source.
  7. In the Ion Source Properties area, input the source settings that have been optimized to allow for high sensitivity and low in-source fragmentation.
  8. TIP

    Copy these settings from Thermo MS Tuning ePanel that is displayed for the system in the Instrument view of the Chromeleon Console.

  9. In the Scan Parameters page, open the list of system templates and select Peptide Mapping > Biopharma High Flow LC template.
  10. In the Time Range box, specify the time window of the first LC gradient.
  11. For example, for the gradient illustrated in the previous section, this window would be 0-70.
  12. Select the Full Scan node and in the dropdown list for Orbitrap resolution select 120.000.
  13. For the AGC Target, select Standard, which corresponds to 100%.
  14. This gives the best linearity for PQA quantitation across different injection volumes.
  15. Select the node that links the full-MS scan with ddMS2 scans and depending on the mode, specify one of the following:
  16. If in the TopN mode, specify an option for the Data Dependent Mode and the number of scans. A recommended value for an SET and for peptide mapping is 3 scans.
  17. If in the TopS mode, specify the maximum cycle time.
  18. Select the filter nodes of the method, one by one, and adjust the settings. A typical method would apply the following:
  19. Monoisotopic Peak Determination of the MIPS filter set to Peptide.
  20. Charge State filter set to include charge states 2-5 or 2-6 (these ranges assume tryptic digests; otherwise charge state 1 might need to be included, as well).
  21. The Dynamic Exclusion filter set to exclude after each time (1) with exclusion duration set to half of the expected LC peak width and with the Excludes isotopes option enabled.
  22. Keep the default ddMS2 parameters as provided by the template.
  23. In summary, a typical setup of the MS instrument for acquiring MS data that is needed for the PQA characterization would look as follows:
Summary of parameter settings for the method comprising of the full-MS and ddMS2 scans
Summary of parameter settings for the method comprising of the full-MS and ddMS2 scans