Mascot, by Matrix Science, is a search engine that can perform peptide mass fingerprinting, sequence query and tandem mass spectra searches. LabKey Server supports using your existing Mascot installation to search an mzXML file against a FASTA database. Results are displayed in the MS2 viewer for analysis.
Modifying Mascot Settings in LabKey Server
For many applications, the Mascot default settings used by LabKey Server are likely to be adequate, so you may not need to change them. If you do wish to override some of the default settings, you can do so in one of two ways:
- You can modify the default Mascot parameters for the pipeline, which will set the defaults for every search protocol defined for data files in the pipeline (see Set a Pipeline Override).
- You can override the default Mascot parameters for an individual search protocol (see Search and Process MS2 Data).
Parameters to the Mascot engine are specified in an XML format. In LabKey Server, the default parameters are defined in a file named mascot_default_input.xml file, at the pipeline root.
When you create a new search protocol for a given data file or set of files, you can override the default parameters. Each search protocol has a corresponding Mascot analysis definition file, and any parameters that you override are stored in this file, named mascot.xml by default.
Note: If you are modifying a mascot.xml file by hand, you don't need to copy parameter values from the mascot_default_input.xml file. The parameter definitions in these files are merged by LabKey Server at runtime.
Configuring MzXML2Search Parameters
You can control some of the MzXML2Search parameters from LabKey Server, which are used to convert mzXML files to MGF files before submitting them to Mascot.
<note type="input" label="spectrum, minimum parent m+h">MIN_VALUE</note>
<note type="input" label="spectrum, maximum parent m+h">MAX_VALUE</note>
These settings control the range of MH+ mass values that will be included in the MGF file.
Using X! Tandem Syntax for Mascot Parameters
You don't have to be knowledgeable about XML to modify Mascot parameters in LabKey Server. You only need to find the parameter that you need to change, determine what value you want to set it to, and paste the correct line into the
Mascot XML section when you create your
MS2 search protocol.
The Mascot parameters that you see in a standard Mascot search page are defined here:
| GROUP | NAME | Default | Notes |
|---|
| mascot | peptide_charge | 1+, 2+ and 3+ | Peptide charge state to search if not specified |
| mascot | enzyme | Trypsin | Enzyme (see /<mascot dir>/config/enzymes) |
| mascot | comment | n.a. | Search Title or comments |
| pipeline | database | n.a. | Database (see /<mascot dir>/config/mascot.dat) |
| spectrum | path | n.a. | Data file | |
| spectrum | path type | Mascot generic | Data format | |
| mascot | icat | off | Treat as ICAT data? (value: off / on) |
| mascot | decoy | off | Perform automatic decoy search (value: off / on) |
| mascot | instrument | Default | Instrument |
| mascot | variable modifications | n.a. | Variable modifications (see /<mascot dir>/config/mod_file) |
| spectrum | fragment mass error | n.a. | MS/MS tol. (average mass) |
| spectrum | fragment monoisotopic mass error | n.a. | MS/MS tol. (monoisotopic mass) |
| spectrum | fragment mass error units | n.a. | MS/MS tol. unit (average mass, value: mmu / Da) |
| spectrum | fragment monoisotopic mass error units | n.a. | MS/MS tol. unit (monoisotopic mass, value: mmu / Da) |
| spectrum | fragment mass type | n.a. | mass (value: Monoisotopic / Average) |
| mascot | fixed modifications | n.a. | Fixed modifications (see /<mascot dir>/config/mod_file) |
| mascot | overview | Off | Provide overview in Mascot result |
| scoring | maximum missed cleavage sites | 1 | Missed cleavages |
| mascot | precursor | n.a. | Precursor |
| mascot | report top results | n.a. | Specify the number of hits to report |
| mascot | protein mass | n.a. | Protein Mass |
| protein | taxon | n.a. | taxonomy (See /<mascot dir>/config/taxonomy) |
| spectrum | parent monoisotopic mass error plus | n.a. | Peptide tol. maximum of plus and minus error |
| spectrum | parent monoisotopic mass error minus | n.a. | Peptide tol. |
| spectrum | parent monoisotopic mass error units | n.a. | Peptide tol. unit (value: mmu / Da / % / ppm) |
| mascot | import dat results | false | Import Mascot search results directly from .dat file (value: true / false) |
The general format for a parameter is as follows:
- <note type="input" label="GROUP, NAME">VALUE</note>
For example, in the following entry, the parameter group is
mascot, and the parameter name is
instrument. The value given for the instrument type is "MALDI-TOF-TOF".
- <note type="input" label="mascot, instrument">MALDI-TOF-TOF</note>
LabKey Server provides additional parameters for X! Tandem for working with the data pipeline and for performing quantitation, described in the following sections.
Pipeline Parameters
The LabKey Server data pipeline adds a set of parameters specific to the web site. Please see Pipeline Parameters section in
Configure X!Tandem Parameters.
Pipeline Prophet Parameters
The LabKey Server data pipeline supports a set of parameters for controlling the PeptideProphet and ProteinProphet tools run after the peptide search. Please see Pipeline Prophet Parameters section in
Configure X!Tandem Parameters.
Pipeline Quantitation Parameters
The LabKey Server data pipeline supports a set of parameters for running quantitation analysis tools following the peptide search. Please see Pipeline Quantitation Parameters section in
Configure X!Tandem Parameters.
Examples
Example 1
Perform MS/MS ion search with the followings: Enzyme "Trypsin", Peptide tol. "2.0 Da", MS/MS tol. "1.0 Da", "Average" mass and Peptide charge "2+ and 3+".
<?xml version="1.0"?>
<bioml>
<!-- Override default parameters here. -->
<note type="input" label="mascot, enzyme" >Trypsin</note>
<note type="input" label="spectrum, parent monoisotopic mass error plus" >2.0</note>
<note type="input" label="spectrum, parent monoisotopic mass error units" >Da</note>
<note type="input" label="spectrum, fragment mass error" >1.0</note>
<note type="input" label="spectrum, fragment mass error units" >Da</note>
<note type="input" label="spectrum, fragment mass type" >Average</note>
<note type="input" label="mascot, peptide_charge" >2+ and 3+</note>
</bioml>
Example 2
Perform MS/MS ion search with the followings: allow up to "2" missed cleavages, "Monoisotopic" mass and report top "50" hits.
<?xml version="1.0"?>
<bioml>
<!-- Override default parameters here. -->
<note type="input" label="scoring, maximum missed cleavage sites" >2</note>
<note type="input" label="spectrum, fragment mass type" >Monoisotopic</note>
<note type="input" label="mascot, report top results" >50</note>
</bioml>
Example 3
Perform ICAT data process.
<?xml version="1.0"?>
<bioml>
<!-- Override default parameters here. -->
<note label="pipeline quantitation, residue label mass" type="input">9.0@C</note>
<note label="spectrum, parent monoisotopic mass error plus" type="input">2.1</note>
<note label="spectrum, parent monoisotopic mass error units" type="input">Da</note>
<note label="mascot, variable modifications" type="input">ICAT_heavy,ICAT_light</note>
<!-- search, comp is optional and result could be slightly different -->
<note label="search, comp" type="input">*[C]</note>
</bioml>
