xQuest is a search program for cross-linked peptides based on MS/MS and isotopic labeling.
It is optimized for the use of isotopically labeled cross-linker (x-linker), but works also (albeit less efficiently) with only a light x-linker.
The search is performed in four steps:
MS2 fragment ions are sorted into "common-ions" and "xlink-ions", based on the isotopical shift.
Peaks that are found in both spectra are considered "common" i.e. they do not have the x-linker attached.
Peaks that are found in the heavy spectrum with a fraction of the isotopic shift are labeled as xlink-peaks. They arise from fragment ions with the crosslinker attached
Search for candidate-peptides in a digested database.
For small databases (<= 50 proteins for web version) all possible peptide:peptide combinations are simply enumerated (default web mode).
For larger databases (<= 1000 proteins for web version) candidate peptides are preselected based on a de-novo step.
Tick the "run in iontag mode" option in "advanced parameters" to search in this mode
combination of each candidate-peptide with each other and with the cross-linker alone (monolinks and loop-links)
Matching against the precursor mass. Note: A high mass accuracy here limits the search space considerably
Scoring against the complete ion series calculated from the x-linked peptide:peptide
Only few special settings are needed to start a search:
The cross-linker used with its mass of monolinks and x-links. You can choose from a preselection of commercially available reagents or define your own
The iosotopic shift (if available)
dta files or tab separated files of MS/MS spectra (light and heavy if available)
a fasta database to search
Mgf format: The mgf parser of the webversion is quite strict in respect to the formatting requirements. Please format your mgfs as in the example mgf file in the "example spectra and databases" section. There also the script "merge.pl" can be downloaded, this script can be used to merge dta files to the correct mgf format. It merges all dta files that can be found in the directory.
An installable version of xQuest is now also availaible from here
You can evaluate your results graphically and you can download a xml-file with all assignments.
For more detail see the publication in Nature Methods