Configuring FragPipe for Scaffold

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.

Note, FragPipe version 19 writes MGF files differently than previous versions. This means that searches may not have spectrum information when loaded into Scaffold. Please use FragPipe 18 if you want to see the spectra in Scaffold.

  • First, make sure to point FragPipe to your local MSFragger installation in the Config tab
  • 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 in the Advanced Output Options. 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 under PSM Validation
  • In the Validation tab, make sure to print decoys and check the Generate Reports box in the FDR Filter and Report section
  • Precursor intensity quantitation can be done using the IonQuant option on the Quant (MS1) tab. The default settings work here
  • Load the results using Scaffold's Prefiltered mode. This will pick up on the Percolator probabilities and they can be used for thresholding
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