'Chromatogram information unavailable' small clinical molecule DIA

support
'Chromatogram information unavailable' small clinical molecule DIA Mus  2020-02-06
 

Hi all
Over the past week, I have been successfully extracting and exporting AUC info of precursor masses from batch replicates (x10) of small molecules to determine precision across 10-13 Calibration points.
I am using DIA and all ion fragmentation (bbCID) mode on a Bruker QTOF - Impact II.
Using precursors and quantifiers and MS/MS fragments as Qualifiers only.

However, yesterday morning, after an autoupdate of skyline to 21.1.0.31, any analysis I attempt is displays the message, ‘Chromatogram Information Unavailable’.
The only other change I have made is to the transition table, updating RT’s for 4 precursors and their heavy isotopic internal standards
No other settings have changed. I cant analyse old data with new update either.
I understand, from the skyline discussion forums that this could be a ‘matching’ issue.
The data is definitely acquired and definitely in the correct datafiles.
I have appended a screenshot for help with diagnosis.

Any advice?
Thank you kindly, in advance!

Mus

 
 
Nick Shulman responded:  2020-02-06
Can you send us your Skyline document and at least one of your raw files?

In Skyline, you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including whatever chromatograms may have been extracted.

You should also send us at least one of your raw files. For Bruker, I think that means you should zip up the entire contents of your ".d" directory and send it to us.

If those two zip files are less than 50MB you can attach them to this support request.

Otherwise, you can upload them here:
https://skyline.ms/files.url

I cannot think of anything that may have changed to make your chomatograms unavailable in Skyline 20.1.
A lot of times chromatograms are missing because none of the MS2 spectra matched the precursors in your document. That does not really apply to the stuff in your screenshot, since all of those are precursors, and they will match any MS1 spectrum.
Since all you have in your Skyline document are precursors, you might want to make sure that you really do have MS1 spectra in your data. Also, you should make sure that you have something other than "None" chosen at "Settings > Transition Settings > Full Scan > MS1 Filtering > Isotope peaks included".

-- Nick
 
Mus responded:  2020-02-07
Thanks Nick, for your prompt response.
I have zipped;
- Two raw files
- Two skyline files (1 from successful analysis and one from unsuccessful analysis)
- one screenshot of successful analysis (pre skyline update)

File name: Chromatogram information unavailable_raw files and supporting docs

My aim is to extract AUC for 8 small molecule precursors. 4 standards and 4 heavy isotope labelled internal standards for determination of precision.
MS1 and bbCID ('all ion fragmentation' ) data was collected.

Greatly appreciate your time and advice. look forward to your assessment.
thanks again
Mus
 
Mus responded:  2020-02-07
 
Nick Shulman responded:  2020-02-07
Mus,

You did not actually attach any files.
If your files are larger than 50MB you should upload them here:
https://skyline.ms/files.url

-- Nick
 
Mus responded:  2020-02-07
sorry Nick. It timed out for some reason. its uploaded now. thanks again
 
Nick Shulman responded:  2020-02-07
Mus,

Thank you for uploading your Bruker data files.

It looks like the problem is that the current version of ProteoWizard (which Skyline relies on to read all sorts of mass spec data) thinks that all of the spectra in those data files are blank. That is, that they have zero mz/intensity pairs in them.
I have verified that the current versions of ProteoWizard SeeMS.exe have this same problem. Older versions of SeeMS.exe can read those files just fine.

I will ask around and see if we can figure out what is going on.

Thanks for reporting this bug.
-- Nick
 
matt.chambers42 responded:  2020-02-21
Thanks for the report. I've committed a fix for this. It'll be in the next Skyline-daily and 20.1 patch.