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Frequently Asked Questions

Sarah Haynes edited this page Dec 8, 2021 · 41 revisions

Q: What are the hardware requirements for MSFragger?

A: This depends on the complexity of the search. In general, we recommend at least 16 GB, but complex closed searches and open searches will require more. Additional variable modifications, semi-enzymatic, and non-enzymatic/non-specific searches will require additional memory and processing cores to maintain very short search times. (We do not recommend performing an open search with non-specific cleavage rules.) Performance scales well with the number of processors. Quantification of Bruker timsTOF data will require at least 32 GB.

Q: Does MSFragger require decoys in the FASTA sequence database?

A: Yes, except if you are using MSFragger inside the PD-Node, please do NOT include decoys. Philosopher/FragPipe can download and format a custom database for you (including the addition of reversed/decoy sequences). See this page for more information about using Philosopher to prepare sequence databases.

Q: Do I need to convert raw MS/MS files to mzML for MSFragger searches?

A: MSFragger/Philosopher can analyze Thermo .raw files, but mzML files are required for TMT/iTRAQ quantification (see this tutorial on converting Thermo .raw to .mzML). Searching and label-free quantification of Bruker timsTOF data can be done through FragPipe. Complete workflow compatibility by file format is shown below.

Q: How can I analyze TMT-labeled data with MSFragger/Philosopher/FragPipe?

A: We currently support TMT 6, 10, 11, and 16-plex, and iTRAQ 4 and 8-plex quantification from the mzML file format. Workflows for each are available in FragPipe. For command line use, see this Philosopher tutorial for a step-by-step example of TMT searching and quantification. The high-throughput Philosopher pipeline function can also be used for large-scale isobaric labeling experiments.

Q: Can I use MSFragger results with Skyline?

A: MSFragger search results from both Thermo Orbitrap data and Bruker timsTOF data can be used with Skyline. See this tutorial on importing validated timsTOF PASEF results into Skyline. Similar steps can be followed to import MSFragger results from Thermo data, see the Skyline documentation for more information.

Q: I got a memory error running MSFragger. What can I do?

A: First make sure a 64-bit (x64) version of Java runtime environment is installed. (The latest version of JRE, which is 64-bit, can be downloaded here.) If you’re using MSFragger in the command line, make sure to specify the appropriate amount of memory given your hardware configuration (e.g. -Xmx32G to use 32 GB). The database splitting option (available in FragPipe) can be used to reduce the size of the in-memory fragment ion index that MSFragger generates. If you still run out of memory, you can reduce the size of the fragment ion index a few ways, 1) use only reviewed sequences in your fasta database, 2) decrease digest_max_length, 3) remove some variable modifications, 4) try a fully-enzymatic search.

Q: What is the difference between "precursor_mass_lower/upper" and "precursor_true_tolerance"?

A: The precursor_mass_lower/upper parameters are the precursor mass boundaries used in search. For example, precursor_mass_lower = -20, precursor_mass_upper = 20, precursor_mass_units = 1 would result in 20 ppm precursor mass tolerance. The default open search precursor mass window is precursor_mass_lower = -150, precursor_mass_upper = 500, precursor_mass_units = 0, or [-150, +500] Da.

The precursor_true_tolerance is used to break ties in open searches and is also used as precursor_mass_lower/upper in the first search (if applicable). For example, if precursor_true_tolerance = 20, precursor_true_units = 1, MSFragger would use precursor_mass_lower = -20, precursor_mass_upper = 20, precursor_mass_units = 1 in the first search no matter what the precursor_mass_lower/upper values were.

Q: Can low resolution (e.g. ion trap) MS/MS data be used in MSFragger?

A: Low resolution MS/MS spectra are suitable for closed searches, but we recommend open searches only be performed on high mass accuracy MS/MS spectra (e.g., acquired in an Orbitrap or high-res TOF).

Q: How should experiments with multiple fractions or experimental groups be handled in FragPipe?

A: See this section of the FragPipe tutorial.

Q: Why do I see 'WARNING: cannot open data file' and 'WARNING: CANNOT correct data file' when running PeptideProphet (with FragPipe or command line philosopher)?

A: These warnings are from PeptideProphet and can be ignored.

Q: Why is the reported number of missed cleavages in a peptide different than I expect?

A: PeptideProphet does not consider a cleavage site missed if it is right next to another cleavage site, e.g. the number of missed cleavage sites in the peptide APTTDEDKK would be changed from 1 to 0 by PeptideProphet.

Q: Why can't I see the MSFragger-PD node after the installation?

A: Please check that (1) you have .Net 4.7 or above installed in your computer, (2) the “MSFraggerPDv2x.dll” file is not blocked by your operating system. To unblock it, right click on the file and select ‘Properties’, then in the ‘Attributes’ section, select ‘Advanced’ and then click ‘Unblock’. Alternatively, you can run unblock-file -path "C:\Program Files\Thermo\Proteome Discoverer 2.4\System\Release\MSFraggerPDv24.dll" (make sure this path is correct) from Windows PowerShell as administrator.

Q: I got an error trying to generate a spectral library with EasyPQP. What can I do?

A: Please check that the appropriate 'MS data type' is selected for your LC-MS files on the 'Workflow' tab of FragPipe. Also make sure you have the latest version of EasyPQP by opening Anaconda Prompt and running these two commands:

pip uninstall --yes easypqp

pip install git+

Q: I got an error when trying to update FragPipe. How can I update the tools manually?

A: If GitHub cannot be reached by FragPipe (due to a firewall or some other restriction), you can install the update manually by following these steps:

  1. Go to:
  2. Download zip files there, save it to the root folder of your FragPipe install.
  3. Unpack the zip (using extract here option of your archiver, so that it maintains the folder structure).
  4. You should now have <FP-install>/tools/xxx.jar file.
  5. Run FragPipe as you would normally, it should now be updated.

Q: I got an error reading Thermo .raw files, what are my options?

A: If an error involving ThermoGrpcServerProcess happens, you may have a firewall or network setting that blocks Thermo raw file reading. If you cannot change the firewall/network settings, we recommend converting .raw files to .mzML.

Q: Can I use a custom enzyme with FragPipe?

A: Yes. On the MSFragger tab in FragPipe, first check that the enzyme is not listed in the 'Protein Digestion'>'Load rules' drop-down. To define a custom enzyme, enter the cleavage rules and set the ‘Enzyme name’ field to ‘nonspecific’. Select ‘ENZYMATIC’ from the ‘Cleavage’ drop-down menu.

Q: What are *.pepindex files, and can I delete them?

A: A *.pepindex file contains the built peptide index. It will be reused if the MSFragger version, FASTA file, and search parameters (e.g., missed cleavages, peptide length range, peptide mass range, variable modifications, etc) are the same, slightly shortening runtime. You can delete them if you want.