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

Training course modules and materials

Learn how to get the best out of Mascot directly from the people who develop and support it.

The training course is organised as several independent topics. We recommend starting with the introductory materials, then proceeding to more in-depth topics. Every topic includes presentation slides suitable for self study or as basis for an in-house training course. The slides and speaker notes are available as PDF files, including links to useful exercises and additional videos and tutorials.

Within each topic, the exercises become progressively more difficult, so are intended to be undertaken in order. If you find an exercise too difficult, first follow the link for hints. If these don’t help, or there aren’t any, and you are really stuck, there may be a link to example search results.

If there are any technical problems accessing or viewing the material or if you have suggestions to improve the course, please email

The materials available on this page describe Mascot Server 2.8, Mascot Distiller 2.8 and Mascot Daemon 2.8.

The classic training course (archive recording, 2014) is available as a webcast course and describes Mascot Server 2.5.

Module: Introduction to database searching using Mascot

Presentation slides and notes: PDF (34 pages, 5.3MB)

  • The three ways to search MS and MS/MS data
  • Peptide Mass Fingerprint
  • Sequence Query
  • MS/MS Ions Search
  • Database searching and spectral library searching

Related content:

Module: Search Parameters

Presentation slides and notes: PDF (35 pages, 4.4MB)

  • A detailed look at each of the search parameters
  • Tips and common mistakes
  • Setting defaults


  1. Simple Peptide Mass Fingerprint: PMF1
  2. Tricky Peptide Mass Fingerprint: PMF2
  3. Difficult Peptide Mass Fingerprint: PMF3
  4. Peptide Mass Fingerprint mixture: PMF4
  5. MS/MS Search of ETD data: MSMS6

Related content:

Module: Sequence Databases

Presentation slides and notes: PDF (29 pages, 4.4MB)

  • Which database to choose
  • SwissProt, Uniprot proteomes
  • NCBIprot, UniRef100
  • Nucleic acid databases
  • Clustering with UniGene
  • Searching whole genomes

Related content:

Module: Modifications

Presentation slides and notes: PDF (44 pages, 8.3MB)

  • Types of modifications
  • Unimod
  • Phosphorylation
  • Modification site analysis
  • Error tolerant searches


  1. Error Tolerant Search: MSMS1
  2. Phosphopeptides: MSMS5
  3. High precursor accuracy: MSMS7

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Module: Scoring & Statistics

Presentation slides and notes: PDF (39 pages, 5.3MB)

  • Different ways of finding a match
  • Probability based scoring
  • Result reports
  • Significance thresholds
  • Assigning peptide matches to proteins
  • False discovery rates
  • Rescoring with Percolator


  1. Target-decoy search: MSMS2

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Module: Very Large Searches

Presentation slides and notes: PDF (35 pages, 6.2MB)

  • Challenges of very large searches
  • Viewing large results reports
  • MudPIT protein scoring
  • Exporting search results


  1. Exporting search results: MSMS3

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Module: Spectral Library Searching

Presentation slides and notes: PDF (28 pages, 1.7MB)

  • Searching a spectral library
  • Integrated FASTA database and library search
  • Enabling predefined spectral libraries
  • Creating spectral libraries from search results

Related content:

Module: Mascot Distiller

Presentation slides and notes: PDF (42 pages, 8.5MB)

  • What is Mascot Distiller?
  • Peak picking
  • Automation
  • Importing search results
  • Calling sequence tags
  • De novo sequencing


  1. PMF: DIS1
  2. MS/MS: DIS2

Related content:

Module: Sequence Queries

Presentation slides and notes: PDF (32 pages, 6.8MB)

  • Standard sequence tag
  • Error tolerant sequence tag
  • seq(), comp() and other qualifiers


  1. Easy sequence tag: SQ1
  2. Tricky sequence tag: SQ2
  3. Distiller Search Toolbox: SQ3

Related content:

Module: Automation with Mascot Daemon

Presentation slides and notes: PDF (42 pages, 5.9MB)

  • Installing and using Mascot Daemon
  • Data import filters
  • Real-time monitor
  • Follow-up tasks
  • Quantitation summary report


  1. Data dependent follow-up task: DM1

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Module: Quantitation

Quantitation – Introduction (12 pages, 1.0MB)

  • Supported protocols
  • Creating and modifying quantitation

Quantitation – MS2 based methods (22 pages, 1.8MB)

  • Quantitation using data in peak lists
  • emPAI
  • Multiplex (isobaric, IPTL)
  • Reporter (iTRAQ, TMT, TMTpro)

Quantitation – MS1 based methods (30 pages, 4.0MB)

  • Quantitation using data from raw file
  • Precursor (SILAC, N15, etc.)
  • Average (label free)
  • Replicate (label free)
  • Fractionation
  • Ion mobility filtering

Quantitation – Reporting (19 pages, 2.4MB)

  • Quantitation reports in Mascot Distiller
  • Quantitation summary in Mascot Daemon


  1. iTRAQ quantitation: MSMS4
  2. SILAC quantitation: DIS3

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Module: Crosslinking

Presentation slides and notes: PDF (26 pages, 1.0MB)

  • Setting up a crosslinking experiment in Mascot
  • Validating intact crosslinks
  • Visualising in xiVIEW

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Module: Sequence Database Administration

Presentation slides and notes: PDF (55 pages, 4.2MB)

  • Sequence database formats
  • Spectral library formats
  • Adding a new database
  • Worked examples
  • Automatic updating


  1. Configure AA Database: ADM1
  2. Configure NA Database: ADM2

Related content:

Module: Administration & Configuration

Presentation slides and notes: PDF (37 pages, 5.0MB)

  • Configuring Mascot Security
  • Mascot configuration files
  • Mascot log files
  • Cluster mode


  1. Configure Mascot Security: ADM3

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