Mascot: The trusted reference standard for protein identification by mass spectrometry for 25 years

What’s new in Mascot Server 3.1

Mascot Server 3.1 is a patch release. It fixes bugs introduced in Mascot Server 3.0 as well as improves the integration with Thermo Proteome Discoverer™.

Resolved: Thermo Proteome Discoverer™ version check

The Mascot node in Proteome Discoverer (PD) queries the product version of Mascot Server to check compatibility. Mascot Server 3.0 responded with “3.0.0″. Unfortunately, PD 3.1 and earlier expects major version number to be “2″, so the version check fails. The main symptom is, you cannot select multiple databases for a Mascot search.

Mascot Server 3.1 now responds with “2.999.0″ if it detects that the client is the Mascot node in PD.

The relevant change was:

  • 14940: Workaround in client.pl?version for Mascot PD node after updating to Mascot Server 3.0

New: Enchanced search results in Thermo Proteome Discoverer™ 1.4, 2.x, 3.x

With Mascot Server 3.1, you can now import results refined with machine learning into Thermo Proteome Discoverer™. Two enhancements have been made:

The Mascot node in PD does not currently have a user interface for enabling refining within Mascot Server. In Mascot Server 3.1, you can now configure an instrument definition that specifies the MS2PIP and DeepLC models to use for refining. Simply select this instrument when you submit the search from PD.

The Mascot node in PD retrieves search results in the dat28 (MIME) format. Mascot Server 3.1 can now encode the refined results in this format, which means all PD versions (1.4, 2.x, 3.x) are now able to import the refined results. This functionality is triggered when a custom instrument definition is used.

The instructions for setting up Mascot Server 3.1 with PD are included in this patch release:

The document also contains an example using a Thermo Orbitrap DDA raw file from PRIDE project PXD028735. We processed this file using Proteome Discoverer 3.1 connected to Mascot Server 3.0 and Mascot Server 3.1. The results are summarised below.

Mascot Server Protein Groups Peptide Groups
3.0 (no refining) 4,635 21,507
3.1 (with MS2PIP:HCD2021 instrument) 5,898 34,141

Full list of relevant changes:

  • 14877: Mascot PD node compatibility: export modified dat28 file with refined results replacing Mascot scores and probabilities
  • 14977: ms_mascotresfile_dat::hasRT() returns false if query 1 has RTINSECONDS[1] or higher index
  • 14990: New option ClientResultFileMimeRefining
  • 14993: Define PERCOLATE and ML_ADAPTER_PARAM in the instrument definition (fragmentation_rules)
  • 14994: Default PERCOLATE and ML_ADAPTER_PARAM from the instrument definition if not submitted as search parameters
  • 15001: Instrument editor should have dropdown menus for MS2PIP model and DeepLC model

Resolved: Minor issues with MS2PIP and DeepLC

The following bugs have been fixed, which caused MS2PIP or DeepLC to fail with certain data sets.

  • 14882: Percolator’s PEP estimator is thrown off by a PSM whose predicted values are all 0 (every PSM gets PEP=1.0)
  • 14934: DeepLC fails to accept ProForma syntax “[Formula:C2 H3 N O]“, caused by e.g. submitting a locally edited copy of a Unimod modification
  • 14942: Want to be able to drop in a custom DeepLC model
  • 14960: Firefox tab freezes when refining a file of 1 million PSMs
  • 14981: ms2pip.exceptions.InvalidPeptidoformError: Peptidoform sequence cannot be longer than 100 amino acids or shorter than 4 amino acids.
  • 14998: When creating MGF input for MS2PIPFeatureGenerator, group spectra by scan number
  • 15009: Update MS2Rescore from v3.0.3 to v3.1.1

Resolved: Failing to filter for variable modification or peptide sequence in Protein Family Summary

The following three bugs have been fixed, which caused issues in the Protein Family Summary report and Report Builder.

  • 14952: Failing to find proteins with a variable modification in MSR file (Report Builder)
  • 14979: Filter unassigned list by ALAGLSPV, fails to find all instances (Unassigned tab)
  • 14980: Failing to find peptides with a variable modification in an MSR file (Proteins tab)

Resolved: Other bug fixes

The patch release also fixes other minor issues, which are listed below and also included in the release notes.

  • 6901: Internal Mascot error (modifications overflow) pl=13, plmax=12 [M00140]
  • 14562: mzIdentML export of decoy report sets isDecoy=false for elements
  • 14753: Wrong sequences in the et_peptides section with an NA database
  • 14866: Remove empty fexpr URL parameters
  • 14891: Ludicrous RAM usage for an ET search when the processor has >= 16 cores
  • 14894: Replace decrypted filename with encrypted filename before displaying refining messages in nph-cache_families.pl
  • 14918: Make Mascot scoring more noise resistant for chimeric spectra
  • 14950: ms-makesearch.log.exe stalls if the directory contains files from crashed searches
  • 14985: client.pl?refine_results should return “not_supported” if a results file cannot be refined
  • 14916: Server error: Class or child modified parameters and did not return them back to previous values
  • 14922: Error: cannot select both Error Tolerant search and Automatic Decoy
  • 14939: Search with looplinked matches outputs bogus modification indices
  • 14984: Flag file is not created when output format is MSR (only affects whether an e-mail would be sent at end of search)
  • 14986: If refining is in progress when calling client.pl?results_header or client.pl?result_file_pop, then the command should poll until refining is done
  • 14999: MS2RescoreReport should be optional, and Protein Family Summary should show a button to create it if not present