Loading Data

File Selection

The first step when using the pyXLMS web application is that you need to upload one or more result files containing crosslink-spectrum-matches (CSMs) or crosslinks/residue pairs (XLs) in the Load Data tab. You can find a list of supported crosslink search engines and input formats here . We also provide some example files via the pyXLMS GitHub repository  that you can try!

Selecting Crosslink Search Engine or File Format

Next you need to select the correct crosslink search engine or file format from the dropdown menu. This should simply be the crosslink search engine that you used for crosslink identification with your mass spectrometry data, or a generic format like mzIdentML.

Selecting Crosslink Reagent

Then you need to select the crosslinking reagent that was used in your experiment from the next dropdown menu. If your reagent is not in the list you can select Custom and two additional input fields will pop up where you can specify a crosslinker name and the monoisotopic delta mass of the crosslink modification.

Reading the Results

If you don’t want to employ any of the additional and optional pre-processing steps (see next section) you can read your results now by clicking on the Read file(s)! button!

[Optionally] Apply One or More Pre-Processing Steps

• Parse Modifications

  Enabling this option will try to parse post-translation-modifications (PTMs) of your CSMs from your result files.

  Note

  Please note that the pyXLMS web app only supports a limited set of the most common PTMs! If parsing fails it is better to leave this option turned off and use the pyXLMS python package directly which gives a more nuanced control over modifications!

• Re-Annotate Crosslink Positions

  This option will allow you to upload a FASTA file to re-annotate the protein crosslink positions of your CSMs and XLs based on the given FASTA file. This might be useful if the annotation is missing because it was not given by the crosslink search engine, or if the crosslink search engine annotated something wrongly.

• Filtering for Unique CSMs and XLs

  If turned on this option will filter out any non-unique CSMs and XLs from your results. A crosslink is considered unique if its sequence and peptide crosslink positions are unique (option 1) or if its protein crosslink positions are unique (option 2) - these options are controlled via the Group crosslinks by selector.

• Validation via FDR Estimation

  Enabling this option will validate your results for a given target false-discover-rate (FDR) via FDR estimation and will only keep CSMs and XLs that fall within the target FDR.

• Filtering for Target Matches

  If applied this option will filter out any CSMs and XLs that are not target-target matches.

• Aggregation of CSMs to XLs

  Whether or not your CSMs (if there are any) should be aggregated to XLs.

• Validation of Aggregated XLs

  Controls if your aggregated XLs from the previous pre-processing step should also be validated via FDR estimation. Shares the same settings as Validation via FDR Estimation.

Reading the Results

You can read your results now by clicking on the Read file(s)! button!