X! Tandem is an open-source search engine that matches tandem mass spectra with peptide sequences. CPAS uses X! Tandem to search an mzXML file against a FASTA database and displays the results in the MS2 viewer for analysis.

Modifying X! Tandem Settings in CPAS

For many applications, the X! Tandem default settings used by CPAS 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 X! Tandem parameters for the pipeline, which will set the defaults for every search protocol defined for data files in the pipeline (see Set Up the Data Pipeline).
• You can override the default X! Tandem parameters for an individual search protocol (see Search and Process MS/MS Data).

X! Tandem takes parameters specified in an XML format. In CPAS, the default parameters are defined in a file named 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 X! Tandem analysis definition file, and any parameters that you override are stored in this file, named tandem.xml by default.

Note: If you are modifying a tandem.xml file by hand, you don't need to copy parameter values from the default_input.xml file. The parameter definitions in these files are merged by CPAS at runtime.

Using X! Tandem Syntax

You don't have to be knowledgeable about XML to modify X! Tandem parameters in CPAS. 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 X! Tandem XML section when you create your MS2 search protocol.

Most X! Tandem parameters are defined in the X! Tandem documentation, available here:

http://www.thegpm.org/TANDEM/api/index.html

As you'll see in the X! Tandem documentation, 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 residue, and the parameter name is modification mass. The value given for the modification mass is 227.2 daltons, at cysteine residues.

• <note type="input" label="residue, modification mass">227.2@C</note>

CPAS provides additional parameters for X! Tandem for working with the data pipeline and for performing quantitation, described in the following sections.

Pipeline Parameters

The following parameters are defined on the pipeline group.

pipeline, database: The path to the FASTA sequence file to search. For example:

• <note label="pipeline, database" type="input">150Proteins.fsa</note>

pipeline, protocol name: The name of the search protocol defined for a data file or set of files. For example:

• <note label="pipeline, protocol name" type="input">ICATSrch</note>

pipeline, protocol description: The description for the search protocol. For example:

• <note label="pipeline, protocol description" type="input">X! Tandem parameters optimized for ICAT labelled samples</note>

pipeline, email address: Email address to notify of successful completion, or of processing errors.

Pipeline Quantitation Parameters

The CPAS data pipeline supports running quantitation analysis tools, through a set parameters passed to X! Tandem. These parameters are defined on the pipeline quantitation group:

residue label mass: Specify this parameter to run quantitation. The format is the same as X! Tandem's residue, modification mass. There is no default value. For example:

• <note label="pipeline quantitation, residue label mass" type="input">9.0@C</note>

algorithm: The default value is "xpress"; other options are not yet implemented.

mass tolerance: The default value is 1.0 daltons.

mass tolerance units: The default value is "Daltons"; other options are not yet implemented.

fix: Possible values "heavy" or "light".

fix elution reference: Possible values "start" or "peak". The default value is "start".

fix elution difference: A positive or negative number.

metabolic search type: Possible values are "normal" or "heavy".

Examples of Commonly Modified Parameters

As you become more familiar with CPAS and X! Tandem, you may wish to override the default X! Tandem parameters to hone your search more finely. Note that the X! Tandem default values provide good results for most purposes, so it's not necessary to override them unless you have a specific purpose for doing so.

The get started tutorial overrides some of the default X! Tandem parameters to demonstrate how to change certain ones. The override values are stored with the tutorial's ready-made search protocol, and appear as follows:

<?xml version="1.0" encoding="UTF-8"?>
<bioml>
 <!-- Override default parameters here. -->
 <note label="spectrum, parent monoisotopic mass error minus" type="input">2.1</note>
 <note label="spectrum, fragment mass type" type="input">average</note>
 <note label="residue, modification mass" type="input">227.2@C</note>
 <note label="residue, potential modification mass" type="input">16.0@M,9.0@C</note>
 <note label="pipeline quantitation, residue label mass" type="input">9.0@C</note>
</bioml>

Taking each parameter in turn:

• spectrum, parent monoisotopic mass error minus: The default is 2.0; 2.1 is specified here to allow for the mass spectrometer being off by two peaks in its pick of the precursor parent peak in the first MS phase.
• spectrum, fragment mass type: The default value is "monoisotopic"; "average" specifies that a weighted average is used to calculate the masses of the fragment ions in a tandem mass spectrum.
• residue, modification mass: A comma-separated list of fixed modifications.
• residue, potential modification mass: A comma-separated list of variable modification.
• pipeline quantitation, residue label mass: Specifies that quantitation is to be performed (using XPRESS).