extract chromatograms from tims TOF

extract chromatograms from tims TOF tleonova  2021-06-18
Dear Skyline team support,

I am quite new to Skyline and I try to use it for MS1 filtering and spectra library building.
I have data acquired on Bruker tims TOF. I used MGF files created by Bruker software for the search in Proteome Discoverer 2.2.

I want to import peptide search from Proteome Discoverer (.msf). But I got stuck at the Extract Chromatograms window. The MGF files were missing.

I found that you already answered to the similar request. I realised that I have to add RAW files for chromatogram extraction. In my case RAW files are .d folders. But when I try to locate these files by clicking on the Find button, navigating to the RAW file location, and clicking these .d folders, nothing happens.

Am I doing something wrong?

Thanks in advance for your help!

Kind regards,
Nick Shulman responded:  2021-06-18
You are supposed to be able to choose .d folders when you are using that "Find" button on the "Extract Chromatograms" page of the Import Peptide Search wizard.

The way that Skyline recognizes that a .d folder is a Bruker raw file is that the folder contains a file named either "analysis.baf" or "analysis.tdf". If the folder does not contain a file with either of those names, then Skyline thinks it is an ordinary folder that happens to have a ".d" at the end of its name, and you will not be able to choose it for chromatogram extraction.

Do the .d folders that you are trying to choose contain a file with either of those names?

-- Nick
tleonova responded:  2021-06-18
Yes, each .d folder contain "analysis.tdf" file.

But when I try to locate these files and click on them, nothing happens.

Attached screenshot shows how it looks like when I click "Find" button.

Nick Shulman responded:  2021-06-18
Can you zip up one (or all) of those .d folders and send it to us?
If that zip file is less than 50MB you can attach it to this support request. Otherwise, you can upload it here:

Can you try copying one of those .d folders to somewhere else on your hard drive and see whether you can use it from that new location? I am wondering whether there is something weird about one of the parent folders or the drive letter where those files are which is messing things up.
A different theory is that maybe Skyline is busy looking in each and everyone one of those .d folders to see if it contains "analysis.tdf", and because Skyline is busy checking the folders, you can't click on anything. If that's the case, then the problem would be solved by copying one of those folders somewhere else where it was the only .d folder.

Can you try opening those files using ProteoWizard MSConvert? The code that Skyline uses to browse for mass spec data files is very similar to, but not exactly the same as, the code that MSConvert uses to do that. You can install ProteoWizard from here:

-- Nick
Brendan MacLean responded:  2021-06-18
Can you include a screenshot of what Skyline is asking you to locate before you bring up the form showing the .d files?

It is important that the names Skyline is asking you for match the names you choose in the form you have shown. That usually requires that your MGF files have matching basenames with your .d folders.

For instance, for the .d folder:


It would normally be important that you used an MGF file with the matching name:


If Skyline is not asking you for something with the basename "1_Slot1-47_1_862" then it will ignore any attempt ot supply this .d folder as what it is looking for. To get things to match up, it may be necessary to rename your .d folders, or redo your search with renamed MGF files. I recently had to do this for MaxQuant results provided with a published paper. For some reason the Thermo .raw files on PRIDE had an added suffix which was not present in the Andromeada search results.

Thanks for posting the screenshot. It always helps to be able to see what you are seeing.

Brendan MacLean responded:  2021-06-18
It seems important to note that for MS1 filtering from DDA, Skyline is attempting to match the peptide IDs in your spectral library with the data files you are providing for chromatogram extraction in order to use the retention times of the MS/MS spectra with peptide IDs. It is not enough to simply supply a set of raw data files which do not match up with your spectral library. This would leave Skyline with much less retention time information to use in alignment, limit the range of chromatogram extraction, and use in peak picking.

That is why we need the basename matching I have described.
tleonova responded:  2021-06-21
Dear Nick, dear Brendan,

I realised where was a problem. Brendan was right, it was an issue with filenames.
All the MGF files inside of my .d folders have the same names as corresponding folders but with suffix "_5.2.216".
Now I added this suffix to the folder names and everything works.

Thanks a lot!
And sorry for taking your time

Kind regards,