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:
For detailed explanation and examples of linear-peptide searching mode, please see references below.
XlinkX performs the search in two steps:
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.
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.
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:
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 .fasta and .mgf files can be downloaded here.
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.