XlinkX/PD
XlinkX/PD is a node in the commercially available Proteome Discover. For more information on how to acquire this platform and the XlinkX/PD nodes see the thermo website.
We receive a small fee for each XlinkX/PD license intended to cover development costs.
Setting up the workflow
Adding the crosslink search to processing workflow is pretty straightforward. You can set up the workflow for a normal peptide search first. After follow these steps:
- Add and connect the node Spectrum Confidence Filter to the peptide FDR control step you selected. This will remove all the spectra that were already confidently identified as a peptide (or PSM) from the crosslink search.
- Add the node XlinkX/PD Detect and connect this to Spectrum Confidence Filter. This is the initializing step where you can set the crosslinking reagent and the used acquisition strategy.
- Add the node XlinkX/PD Search and connect this to XlinkX/PD Detect. This is where the search takes place and you can set here things like the Protease, modifications, etc.
- Add the node XlinkX/PD Validate and connect this to XlinkX/PD Search. Here validation of the identification happens at the spectral level (CSM).
With this setup, the software will be able to make optimal use of the normal peptide search and get the best results for your crosslinked peptide pairs.
Likewise, you can extend the normal consensus workflow in the following manner:
- Add the node XlinkX/PD Crosslink Grouping and connect this to Protein Grouping. After the protein grouping is finalized, all information from the normal peptide search has been deposited allowing the grouping to not only role multiple CSMs into a single Crosslink entry, but also to connect the entries to the proteins.
- Add the node XlinkX/PD Consensus Validator and connect it to XlinkX/PD Crosslink Grouping. At this point the final FDR steps are taken to remove false positives at the crosslink level. This table will have a higher false positive rate without this step, as false positive CSMs tend to exist in isolation while true positive CSM tend to come in multiples. As these multiples are grouped in a single Crosslink entry, this can increase the false positive rate in this table.
- If so desired, connect the node XlinkX/PD Crosslink Export to XlinkX/PD Consensus Validator. This node will automatically export the results to a pre-determined location.
Defining a new crosslinker
Defining your own crosslinking reagent can be done in the Modifications tab in Proteome Discoverer.
- Open the Chemical Modifications tab;
- Double click a modification -or- press Add in the menu right above;
- Use the dialog to make the required settings;
This supports all linkers (incl. cleavable and non-cleavable).
