After you defined the LC part of the instrument method, configure the MS detection.
Prerequisites
- The MS instrument is installed and configured in Chromeleon CDS software using a valid license.
- 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
- In the Method Editor, set the Application Mode to Peptide.
- Set the Method Duration to match the run time that you specified in the LC part of the instrument method.
- In the Settings area > Infusion Mode, select Liquid Chromatography and enable Advanced Peak Determination.
- This provides better determination of peptide charge states and monoisotopic m/z values of isotopic envelopes. Keep the Default Charge State equal to 2.
- 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.
- In the Global Parameters tab, select Ion Source.
- In the Ion Source Properties area, input the source settings that have been optimized to allow for high sensitivity and low in-source fragmentation.
TIP
Copy these settings from Thermo MS Tuning ePanel that is displayed for the system in the Instrument view of the Chromeleon Console.
- In the Scan Parameters page, open the list of system templates and select Peptide Mapping > Biopharma High Flow LC template.
- In the Time Range box, specify the time window of the first LC gradient.
- For example, for the gradient illustrated in the previous section, this window would be 0-70.
- Select the Full Scan node and in the dropdown list for Orbitrap resolution select 120.000.
- For the AGC Target, select Standard, which corresponds to 100%.
- This gives the best linearity for PQA quantitation across different injection volumes.
- Select the node
that links the full-MS scan with ddMS2 scans and depending on the mode, specify one of the following:
- 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.
- If in the TopS mode, specify the maximum cycle time.
- Select the filter nodes of the method, one by one, and adjust the settings. A typical method would apply the following:
- Monoisotopic Peak Determination of the MIPS filter set to Peptide.
- 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).
- 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.
- Keep the default ddMS2 parameters as provided by the template.
- In summary, a typical setup of the MS instrument for acquiring MS data that is needed for the PQA characterization would look as follows: