XlinkX: a dedicated search engine for linked peptides

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Introduction

XlinkX is a dedicated algorithm that searches disulfide-bridged and cross-linked peptides. It operates in two searching modes, enumeration mode and linear-peptide mode. In enumeration mode, The cross-link is searched by considering all possible peptide-peptide combinations for the cross-link precursor mass. As the database size enormously increases with the number of peptide in the database, it is recommended to use a database containing less than 50 proteins. In the linear-peptide mode, the cross-linked peptide is searched by obtaining the precursor mass of each linked peptide based on the signiture fragmentation pattern of the cross-link, it therefore is able to search against proteome databases. To use linear-peptide mode, the input spectra need to satisfy the following requirements:

  1. In disulfide-bridged peptide identification, ETD-based MS2 spectra (ETD and EThcD) are required.
  2. In cross-linked peptide identfication, MS2 spectra originated from MS-cleavable cross-linkers are required. In the current XlinkX version, CID MS2 spectra of DSSO (CID cleavable) cross-linker and ETD (EThcD) spectra of DTSSP (ETD-cleavable) cross-linker are supported.

For detailed explanation and examples of linear-peptide searching mode, please see references below.


Perform the search

XlinkX performs the search in two steps:

1. Index database

The fist step is to prepare a database for the main search. One can either choose a pre-existing database or upload a fasta file to index a database. We recommend the uploaded fasta file do not exceed a protein number of 1000. After filling in all required parameters, click the "Submit" button on the bottom of the page.

2. Main search

After preparing databases, the main search can be performed. XlinkX works the best with high-resolution LC/MS data on both MS and MS2 levels. In the current version, XlinkX only takes mgf files as input. mgf files can be generated by software such as Proteome Discover (Thermo Fisher Scientific). For detailed instructions on how to acquire data and prepare mgf files, see references below.

Search result

1. Table of cross-link

When the search finishes, a result table containing all unique residue-to-residue cross-links is presented. Part of the result file is shown below as an example.



Column headers are described as follows:

  1. index, the software generated number for spectra annotation.
  2. file_name, the name of the searched mgf file.
  3. scan, the scan number.
  4. charge, the charge state of the precursor.
  5. precursor mass, the monoisotopic neutral mass of the precursor.
  6. spectra_num, the PSM number of the unique cross-link identification.
  7. peptide_a/peptide_b, the peptide a/b sequence.
  8. xl_a/xl_b, the cross-linked residue number of the peptide.
  9. pep_pos_a/pep_pos_b, the location of the peptide in the protein.
  10. mass_a/mass_b, the monoisotopic neutral mass of the peptide.
  11. match_a/match_b, the number of matched fragment ions.
  12. n_score_a/n_score_b, the n_score of peptide a/b.
  13. match_ttl, the total number of matched fragment ions, which is the sum of match_a and match_b.

2. Spectra annotation

Annotated spectra can be retrieved in the above spectra labeling tab. By submitting the index number of each cross-link, a annotated spectrum will be presented. An example annotated spectrum from linear-peptide search mode is shown here.

An example annotated spectrum from enumaration search mode is shown here.

Example files

Example .fasta and .mgf files can be downloaded here.

Contact us

XlinkX is developed by Fan Liu in the lab of Albert J.R. Heck. Special thanks to Bas van Breukelen and Henk van den Toorn for the assistance of setting up the online web tool. For more information and inquiries, please contact Albert J.R. Heck.

References

  1. Liu, F., Breukelen, B. Van & Heck, A. Facilitating protein disulfide mapping by a combination of pepsin digestion, Electron Transfer higher energy Dissociation (EThcD) and a dedicated search algorithm SlinkS. Mol. Cell. Proteomics 1-33 (2014).
  2. Liu, F., Rijkers, D, Post H., Heck, A., Structural profiling of endogenous protein assemblies and interactions in human cell lysates by cross-linking mass spectrometry. (Submitted)

Parameters


Modifications

Fixed modifications
Variable modifications Maximum oxidation (M) number per peptide
Maximum phosphorylation (S,T,Y) number per peptide

            

Parameters

the n-score value is the 10th power of the input value (e.g. an input of -6 equals to an n-score of 10^(-6)
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Index number