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If the crosslinker is specified in the MS Annika Search Node as a fixed or variable modifications (in addition to specifying it as the crosslink modification in the MS Annika Detector Node), an error is thrown. This is intended behaviour because in (almost) all cases this is not what you want. The modifications happening to the two cross-linked peptides during cross-linking are already accounted for by the setting in the MS Annika Detector Node.

For the rare case that you actually additionally want to consider that these peptides might carry an additional crosslinker on one of the other residues (e.g., as a monolink modification) you would have to duplicate the crosslinker in the "Chemical Modifications" window in Proteome Discoverer and give it another unique name and select this one instead. That should solve the issue and the search will run through completely.

• Due to the limitations of our matrix search it is recommended to set a maximum peptide length of less than 50
• For small databases this doesn't matter
• For big databases this matters a lot, peptides longer than 50 amino acids trigger re-allocations of the sparse matrix which are very costly and a search that would take 30s suddenly takes 20min
• Going with the default maximum peptide length in MS Annika is usually a good choice!

The current MS Annika 2.0 v2.2.1 / v1.2.0 installer doesn't work with the newest release version of Proteome Discoverer 3.1 (v3.1.0.638).
It's recommended to stay on Proteome Discoverer 3.0 for the moment until this is fixed!

In Proteome Discoverer 3.1 using the Protein Scorer node in the consensus workflow will yield an error with MS Annika. This error does not terminate the search (or the creation of the result) and does not affect crosslink results!

However, as a result it currently might not be possible to calculate Protein FDR with MS Annika in Proteome Discoverer (in case that would be something you are interested in - we don't have experience with that).

We recommend using xiFDR in combination with our xiFDR exporter for higher-level FDR calculations.
Nevertheless, we are working on a fix!

In some rare cases (usually when the FASTA headers are not in a standard format) a Proteome Discoverer MS Annika 2.0 job might be interrupted by the following assertion warning. If it pops up just press "Ignore" and the job will complete successfully. This issue only happens in Proteome Discoverer version <= 3.0 / MS Annika 2.0 version <= 1.1.4

Proteome Discoverer doesn't read scan numbers from MGF files correctly and just numbers spectra from 0 to n. This messes up down-stream analysis as CSMs can't be properly mapped back to the spectra they originate from. This can't be fixed in MS Annika as MS Annika doesn't read the MGF file, Proteome Discoverer does - however it is possible to remap output files with the this tool we implemented: https://github.com/hgb-bin-proteomics/Proteome_Discoverer_MGF_Scan_Number_Repair_Tool

If multiple fasta files are selected for search in MS Annika, users have to make sure that they don't overlap - so do not contain the same protein twice (or more often). Duplicate proteins may lead to a crash in MS Annika as Proteome Discoverer enforces that proteins must be unique. A good practice is to merge fasta files beforehand (so pasting all sequences into one fasta file) and only use one fasta file for search in MS Annika, as Proteome Discoverer makes sure that each fasta file only contains unique proteins.