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Processing files for histone PTM analysis using .d files

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Processing files for histone PTM analysis using .d files lorenz donndorf  2025-08-13 02:39
 

Dear support team,

Recently I sent trypsin-digested propionylated mouse histones for mass spectrometry using different treatments, and want to ultimately quantify if I can see changes in any/certain histone PTMs (Like H3K27me3) between my treatments. Additionally, some samples were propionylated using deuterium-labelled propionic anhydride, and the peptides should therefore have a slightly changed mass. The mass spec was run on a Bruker machine and I have DIA .d files.

After having now received these .d files, I am not entirely sure how to process them. As far as I understand, I have to create a spectral library first, and I looked into the "Skyline Targeted Method Editing" tutorial, but in the example provided there the library is not built using .d files, but instead using a prep.xml file. Checking online, I found previous questions on this support forum that were asking how to directly load .d files, but it was mentioned that SkyLine is not intended for viewing raw mass-spec data.

When checking how to make this spectral library using my .d files, I found a tutorial that used the software DIA-NN to generate the spectral libraries using the .d files and calling/running it against a fasta file of the proteins that one is interested in (In my case just histones).

Is this the correct approach, or how should I (pre-)process my .d files to be able load them in SkyLine and be able to diffentiate between histone PTMs and to quantify differences between samples?

Thank you!
 
 
Nick Shulman responded:  2025-08-13 08:43
The "Targeted method editing" tutorial is probably only useful if you have a triple quadrupole instrument.
The tutorials in the "Full Scan Acquisition" sections would probably be better for learning about what to do with DIA methods:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorials

Here is the list of peptide search result formats that Skyline understands:
https://skyline.ms/wiki/home/software/BiblioSpec/page.view?name=BlibBuild

If you have peptide search results in one of those formats, then you can get them into Skyline by doing "File > Import > Peptide Search" which will guide you through creating the spectral library (i.e. a .blib file), importing a FASTA with the list of proteins you are interested in, and extracting chromatograms from your .d folders.

I do not see "prep.xml" in the list of supported search result formats, but you might have some other search results from earlier in your process that BiblioSpec would understand.

Alternatively, if your raw data does not use ion mobility, then you could have Skyline search your results using the menu item:
File > Search > Run Peptide Search.
There is an example of running a peptide search in Skyline in this tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_dda_search

If your Bruker data does use ion mobility then we recommend using MSFragger outside of Skyline. With Bruker ion mobility data, you must use a version of MSFragger which is 4.2 or greater. Unfortunately, Skyline only knows how to run MSFragger 4.1, so you have to run your MSFragger search outside of Skyline and then import the results that you get using "File > Import > Peptide Search".

It sounds like you might also be doing complicated things with PTMs.

Here is a great webinar for learning about working with modifications in Skyline:
https://skyline.ms/Webinar10.url

-- Nick