Bruker timsTOF Support

[xLinkKnight]

Curious to know if timsTOF support might be added to xiSEARCH. The inclusion of the ion mobility dimension might benefit some of the crosslink experiments we are currently performing.

As it is now, xiSEARCH seems to work with Bruker files which are converted to the MGF format using MSConvert. MGFs created using the supplied script in the Bruker DataAnalysis program fail to process in xiSEARCH.

May I also ask if the following would be the correct entry for the CDI crosslinker? I'm pretty sure the inclusion of the LINEARMODIFICATIONS should be removed.
CDI=crosslinker:SymetricSingleAminoAcidRestrictedCrossLinker:Name:CDI;MASS:25.979265;LINKEDAMINOACIDS:K(0),S(0),T(0),Y(0),nterm(0);LINEARMODIFICATIONS:NH2,17.026549105,OH,18.0105647,loop,0;STUBS:Urea,25.979265,Free,0.00

Is it possible to create an enzyme entry for Trypsin/Asp-N(D)? More specifically, can the ConstrainingAminoAcids term be assigned to one but not both enzymes?

Thank you. We would be happy to help if it's of interest.

[lutzfischer]

We used timsTOF data with xiSEARCH before. Can you post the error log? - Or maybe the an example file (could be e.g. just the first two scans of the generated MGF).

Not a chemist and not used CDI before, so take this with a grain of salt, but I think the mass is wrong (was looking it up here: https://www.covachem.com/cdi-carbonyldiimidazole-530-62-1.html). If that is the right crosslinker then I think it should rather have a mass of -17.002739665 (losing one oxigen from D or E and losing one hydrogen from K) as the mass given here is the difference between two peptides on their own and the two peptides linked by the crosslinker.

To my understanding the definition should look something like this:

crosslinker:AsymetricSingleAminoAcidRestrictedCrossLinker:Name:CDI;MASS:-17.002739665;FIRSTLINKEDAMINOACIDS:D,E;SECONDLINKEDAMINOACIDS:K,S,Y,T,nterm;STUBS:Urea,25.979265,Free,0.00

I am not sure about crosslinker modifications - you probably need to talk to an chemist about that.
About the double digest - xi actually has an inbuild mechanism to model that

digestion:MultiStepDigest:consecutive|S|PostAAConstrainedDigestion:DIGESTED:R,K;ConstrainingAminoAcids:P;NAME=Trypsin;MISSEDCLEAVAGES:2|S|PostAAConstrainedDigestion:DIGESTED:D;ConstrainingAminoAcids:;NAME:ASP-N(D);MISSEDCLEAVAGES:2

instead of consecutive you can also use full or independent

• full : every enzyme digests the protein plus the peptides of previous enzymes
• consecutive: every enzyme only digests the peptides of the previous enzyme
• independent: every enzyme only digests the original sequences

defining the MISSEDCLEAVAGES for each enzyme is optional - if all use the same number of missed cleacages then you can just use the global setting.

[xLinkKnight]

Thank you for the thorough explanation. I greatly appreciate the flexibility designed into the program to handle various digestion conditions and crosslinkers.

I was actually following the CDI reaction mechanism which generates a urea (NH2-HN2) or carbamate (NH2-OH) product.
(https://onlinelibrary.wiley.com/doi/full/10.1002/anie.201708273)
The reaction of CDI with an amine and hydroxyl group will generate a characteristic 26-u doublet when fragmented. Please let me know if my interpretation is incorrect.

I've attached the sample MGF files from MSConvert (works with xiSEARCH) and the Bruker script generated MGF (fails).
Sample_MGF_BrukerConverted.txt
Sample_MGF_MSConvertConverted.txt
This might be a moot issue as xiSEARCH can still process timsTOF data. Just wish for an easier way to access the IM information within the xlink results.

[MNTsnowman]

Hi xLinkKnight

I'm not sure if you ave solved the issue or not. However, I have data from a timsTOF as well. I use proteowizard to convert the *.d files into *.mgf files. Make sure to tick the "Combine ion mobility scans" and it should work, atleast it does for me. Also, if you want smaller files you could only allow for top 200 most abundant peaks in the output, but that's optional.