You can use the LabKey data pipeline to initiate a search for peptides on MS/MS data. The search results are displayed in the MS2 viewer, where you can evaluate and analyze the processed data.

To experiment with a sample data set, see the Proteomics Tutorial guide and the Proteomics demo project.

Select the MS/MS Data File

To select a data file to search, follow these steps:

  • First set up the pipeline root. See Set a Pipeline Override.
  • Click the Process and Import Data button.
  • Navigate through the file system hierarchy beneath the pipeline root to locate your mzXML file.
Describe the mzXML File (Optional)

You can optionally create an experiment protocol to describe how the sample was processed and what experimental procedures were used in creating the mxXML file.

  • On the MS2 Dashboard click New Assay Design in the Assay List web part.
  • Select Mass Spec Metadata and specify the current folder as the location unless you want your protocol available more widely.
  • Name your new protocol.
  • Specify the appropriate properties and fields. Options including inferring fields from a sample file can help.
  • Click Save & Close.

You may also want to show a button for your new protocol on the pipeline import toolbar, to make it easier for others to find and use it.

  • Click Data Pipeline > Process and Import Data.
  • Click the Admin button on the toolbar.
  • In the Describe Samples section, click the box for Show on Toolbar for your newly defined protocol.
  • Click Submit.

Start a Search

To search, check the box for the subject .mzXML file and click X!Tandem Peptide Search.

If you have configured Mascot or Sequest, you should see additional buttons to initiate searches for those search engines.

Create a Search Protocol

Next, you need to specify a search protocol. You can create a new search protocol or specify an existing one. If you're using an existing protocol, you can just select it from the Analysis Protocol list. This list shows the names of all protocols that were created for the MS2 search runs that share the same pipeline root and that use the same search engine.

If you're creating a new search protocol, you need to provide the following:

  • A name for the new protocol.
  • A description.
  • A FASTA file to search against. The FASTA files listed are those found in the FASTA root that you specified during the Set a Pipeline Override process.
    • For XTandem and Mascot searches, you can select multiple FASTAs to search against simultaneously.
  • Any search engine parameters that you want to specify, if you wish to override the defaults.
Once you've specified the search protocol, click the Search button to initiate the search. You'll be redirected to the Portal page, where you'll see the search status displayed as the file is processed. Once the status reads COMPLETE, the search is finished.

Note: Large runs can take hours to process. By default, LabKey will run the X! Tandem searches on the same web server where LabKey is running. Mascot and Sequest searches will be run on whatever server is configured in Site Settings. TPP processes (Peptide Prophet, Protein Prophet, and XPRESS quantitation, if configured) are run on the web server by default, for all search engines. If you use LabKey Server to frequently process large data sets, you may want to set up your search engine on a server cluster to handle the load. If you wish to do this, you are using LabKey Server in a production setting and require commercial-level support for cluster set-up. For further information on commercial support, you can contact the LabKey Corporation technical services team.

Search Engine Parameter Format

LabKey Server uses an XML format based on the X! Tandem syntax for configuring parameters for all search engines. You don't have to be knowledgeable about XML to modify search 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 X! Tandem XML section (or Sequest XML or Mascot XML) when you create your MS2 search protocol.

The general format for a search 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>

LabKey Server uses the same parameters across all search engines when the meaning is consistent. The example above for "residue, modification mass" is an example of such a paramter. For these parameters, you may want to refer to the the X! Tandem documentation in addition to the LabKey Server documentation. The X! Tandem documentation is available here:

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

The following sections cover the parameters that are the same across all searches, as well as the specific parameters that apply to the individual search engines:

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