Showing Chromatogram information unavailable after importing results mnissa  2023-01-30 10:45
 

Dear Support Team,
When I import the result files, while looking at the results, it seems as if the whole target list is not at all monitored in all the samples and it shows "Chromatogram information unavailable". However, I have used the same method to run all the samples.
When i checked the raw file, it is ok and have been monitored for all the precursor masses. I tried to import the results in a new Skyline document, some of the files which didn't show up before, they showed up and I could see the data for all the precursors in the target list. But the issue was still there if I remove those files or reimport.
As i have shown in this attachment, the peak is being shown for only sample 1 and not for sample 2-4. If I try starting the whole thing again in a new document It can happen the next time that I see the results for sample 3 or 2 or 4 and not for sample 1. So, it is just happening randomly. As this is not happening to a single or same set of raw files each time, I am not able to guess the issue.
Kindly help me fix this.

Best regards
Mehar
 
 
Nick Shulman responded:  2023-01-30 10:51
Can you send us your Skyline document and a few of your raw files? We will figure out what is going on.

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

If that .zip file and your raw files are less than 50MB you can attach them to this support request. You can upload larger files here:
https://skyline.ms/files.url

I cannot tell whether your data was SRM or chromatograms extracted from full spectra.
For SRM, the usual reason that chromatograms are "unavailable" is that there were not chromatograms in the raw file whose Q1 and Q3 values matched the precursor and transition m/z's in your document.
For full scan, the usual reason is that the MS/MS Acquisition settings at "Settings > Transition > Full Scan" did not result in MS2 spectra matching any of the precursors in your document.

We will probably be able to figure out what is going wrong after we see your files.
-- Nick
 
mnissa responded:  2023-01-30 13:03
Dear Nick,
Thanks for your kind response.
I have shared the Skyline document and a few raw files for your perusal.
Two zip folders are uploaded (here https://skyline.ms/files.url) named as under:
20230130_Mehar_raw
20230130_Mehar_Skyline

I look forward to your response.

Best regards
Mehar
 
Nick Shulman responded:  2023-01-30 15:04
Thank you for sending those files.

You should go to:
Settings > Transition Settings > Full Scan
and select "Include all matching scans" in the "Retention time filtering" box.

The problem is that you have chosen "Use only scans within 5 minutes of MS/MS IDs", and the times of the PSMs in your spectral library are not close to the times of the spectra that you want to extract chromatograms from.

With PRM data, you nearly always want to choose "Include all matching scans" because you have already told the mass spectrometer what times to collect spectra for your peptides, and you would want Skyline to use all of those PRM spectra.

After you have changed your transition settings you can tell Skyline to extract chromatograms again by going to "Edit > Manage Results > Reimport".
-- Nick