When setting up FragPipe for Scaffold the defaults for closed searches should work fine. Or these can be used as a starting point and can be edited from there. For general FragPipe information see their documentation There are a few points to be mindful of. See this documentation for running MSFragger through Scaffold as opposed to through FragPipe.
- In the MSFragger tab, make sure the write calibrated MGF box is clicked in the Advanced Output Options.
- Leave the Output format dropdown set to PEPXML_PIN. Changing this can cause issues when trying to load the data in Scaffold.
- In the Validation tab, disable Crystal-C
- In the Validation tab, make sure to run Percolator and check the Run PSM Validation box
- In the Validation tab, make sure to print decoys and check the Generate Reports box
- Run Scaffold in Prefiltered mode. This will pick up on the Percolator probabilities and they can be used for thresholding.
- Precursor intensity quantitation can be done using the IonQuant option on the MS1 Quantitation tab. The default settings work here.