Why are Batch and Manual Processing not providing the same results?

In order to produce similar results with manual and automatic processing, be sure to sum the exact same spectra and use the same spectra for background subtraction.

If large differences are still observed there is a setting in the processing method file that may need to be adjusted, depending on which version of ProMass is being used.

ProMass for Xcalibur version 5 users: If the default mass tolerance settings in the processing method are used (500 mmu), ProMass will automatically set an optimum value for the mass tolerance based on the instrument resolution setting in the raw file. If you want to override the ProMass optimized setting, you can set a ppm value, as described below. This is only applicable to high resolution (e.g., Orbitrap) data.

ProMass for Xcalibur version 3 and 4 users: The mass tolerance setting in your processing method should be optimized for your instrument resolution setting. Open your Xcalibur processing method in the Processing Method Setup program. The mass tolerance setting can be found under Options -> Masses. This setting only needs to be adjusted for Orbitrap data sets.

This will open the dialog box shown above. For Orbitrap data, changing the Mass tolerance ppm value will allow you to more accurately match the results obtained by batch processing to those obtained by manual processing. Use the following as a guide:

• For ion trap or quadrupole MS data, use the default setting of 500 mmu.

• ProMass for Xcalibur version 5 users should use the default setting of 500 mmu to have ProMass automatically apply optimized settings based on the instrument resolution in the data file.

ProMass for Xcalibur version 3 or 4 users should use the following guide:

• For isotopically unresolved Orbitrap data, use a value of 20-100 ppm.

• For isotopically resolved Orbitrap data, use a value of 1-10 ppm, depending on your instrument resolution setting. The following formula can be used as a starting point to optimize the mass tolerance of isotopically resolved data:

mass tolerance (ppm) = C / R

• where C is a constant dependent on the instrument used (C = 200,000 for the older LTQ Orbitrap series instruments and C = 300,000 for Q-Exactive, Fusion, and later instruments). R is the instrument resolution setting used for the data acquisition. As an example, a resolution setting of 60,000 on an Orbitrap Fusion should use a mass tolerance of 5 ppm in the processing method.

Even after optimization, it is still possible that there will be slight differences between results for manual and batch processing. This is due to the differences in averaging spectra in Xcalibur Qual Browser and the processing method files. For more information, see the article entitled: Setting the Correct Mass Tolerance in the Xcalibur Processing Method for ProMass HR Operation.