Sequest, by Thermo Sciences, is a search engine that matches tandem mass spectra with peptide sequences. LabKey Server uses Sequest to search an mzXML file against a FASTA database and displays the results in the MS2 viewer for analysis.
Because LabKey Server can search with several different search engines, a common format was chosen for entering search parameters. The format for the search parameters is based on the input.xml format, developed for X!Tandem. LabKey Server includes a set of default Sequest search parameters. These default parameters can be overwritten on the search form.
Modify Sequest Settings in LabKey Server
Sequest settings are based on the sequest.params file (See your Sequest documentation). For many applications, the Sequest 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:
- Modify the default Sequest parameters for the pipeline, which will set the defaults for every search protocol defined for data files in the pipeline. Learn more in this topic: Set a Pipeline Override.
- Override the default Sequest parameters for an individual search protocol. Learn more in this topic: Search and Process MS2 Data
Sequest takes parameters specified in XML format. In LabKey Server, the default parameters are defined in a file named sequest_default.input.xml, 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 Sequest Sequest analysis definition file, and any parameters that you override are stored in the file, named sequest.xml by default.
Note: If you are modifying a sequest.xml file by hand, you don't need to copy parameter values from the sequest_default_input.xml file. The parameter definitions in these files are merged by LabKey Server at runtime.
Configuring MzXML2Search Parameters
The mzXML data files must be converted to Sequest .dta files to be accepted by the Sequest application. The MzXML2Search executable is used to convert the mzXML files and can also do some filtering of the scans that will be converted to .dta files. Arguments are passed to the MzXML2Search executable the same way that parameters are passed to Sequest. The available MzXML2Search parameters are:
|-F<num>||MzXML2Search||first scan||none||Where num is an int specifying the first scan|
|-L<num>||MzXML2Search||last scan||none||Where num is an int specifying the last scan|
|-C<n1>[-<n2>]||MzXML2Search||charge||1,3||Where n1 is an int specifying the precursor charge state to analyze and n2 is the end of a charge range (e.g. 1,3 will include charge states 1 thru 3).|
Using X!Tandem Syntax for Sequest Parameters
You don't have to be knowledgeable about XML to modify Sequest parameters in LabKey Server. You only need to find the parameter that you need to change, determine the value want to set it to, and paste the correct line into the Sequest XML section when you create your MS2 search protocol
When possible the Sequest parameters will use the same tags already defined for X!Tandem. Most X!Tandem tags are defined in the X!Tandem documentation, available here:
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="residue, modification mass">227.2@C</note>
LabKey Server provides additional parameters for Sequest where X!Tandem does not have an equivalent parameter, for working with the data pipeline and for performing quantitation, described in the reference topic: sequestParams}
Examples of Commonly Modified Parameters
As you become more familiar with LabKey proteomics tools and Sequest, you may wish to override the default Sequest parameters to hone your search more finely. Note that the Sequest 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 [Discover Proteomics Tutorial overrides some of the default X! Tandem parameters to demonstrate how to change certain ones. Below are the override values to use if Sequest is the search engine:
<?xml version="1.0" encoding="UTF-8"?>
<!-- Override default parameters here. -->
<note label="spectrum, parent monoisotopic mass error plus" type="input">2.1</note>
<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>
<note label="pipeline quantitation, algorithm" type="input">xpress</note>
Taking each parameter in turn:
- spectrum, parent monoisotopic mass error minus: The default is 2.0; 2.1 is specified here. Sequest requires a symmetric value so both plus and minus must be set to the same value.
- 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 the residue and weight difference for quantitation.
- pipeline quantitation, algorithm: Specifies that quantitation is to be performed (using XPRESS).
The Sequest/LabKey Server integration was made possible by: