Chromatogram not loading

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Chromatogram not loading prajita  2022-03-17
 

I am trying to do some MS1 filtering to determine my precursor ions. I imported all FASTA sequence from the proteins. I also imported the result which is my raw file. The software is saying TIC not available. I have done this in the past and it has worked. Can you please help me with this?

 
 
Nick Shulman responded:  2022-03-17
If you are seeing the message "No TIC chromatogram found", it means that Skyline is trying to show you the total ion current chromatogram, but that chromatogram was not extracted.
You probably want to be looking at the chromatograms for the currently selected chromatogram. You should right-click on the chromatogram graph and choose "Transitions > All".

When you import results, Skyline tries to also extract the total ion current chromatogram, because that chromatogram can be informative to look at, and Skyline can use that chromatogram for normalization if you choose "Total Ion Current" as the normalization method at "Settings > Peptide Settings > Quantification".
-- Nick
 
prajita responded:  2022-03-17
I made all those changes but it still says unavailable. I have attached a screenshot.
 
Nick Shulman responded:  2022-03-17
Can you send me your Skyline document and your raw file?
In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files.

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

Does your raw file have SRM data in it, or were you extracting chromatograms from full scan MS1 and MS2 data?
With SRM data, the usual reason to see the "Chromatogram information unavailable" message is that there were no SRM chromatograms in the file which matched the precursor and product m/z's of the transitions in your document. When I want to figure out why that is happening, I use ProteoWizard SeeMS.exe to look at the raw file. If the difference between the m/z's in the raw file and in your Skyline document are small, then you can usually fix this problem by choosing a larger number for "method match tolerance m/z" at "Settings > Transition Settings > Instrument".
With full scan data, the problem is usually that there were no MS2 spectra which matched the precursors in the document. I usually use SeeMS.exe to diagnose this sort of problem too, and maybe adjust the settings at "Settings > Transition Settings > Full Scan".
-- Nick
 
prajita responded:  2022-03-17
Please find the attached file.

I was extracting chromatograms from full scan MS1 data.
 
Nick Shulman responded:  2022-03-17
Can you zip up and send me one of your .d folders?

The instrument info in the Skyline document seems to think that those came from an Agilent triple quad machine.
-- Nick
 
prajita responded:  2022-03-17
It did come from Agilent triple quad.
 
prajita responded:  2022-03-17
I also wanted to send you the one that worked. They came from the same instrument. Its uploaded to the link you had sent.
 
Nick Shulman responded:  2022-03-17
I see that the "MatrixBlank-01.d" is much smaller (less than a megabyte) than the other file that you uploaded "Blank-03.d" (128 megabytes).
MatrixBlank-01.d does not have any MS1 spectra in it, which is why Skyline does not extract any chromatograms for you.

MatrixBlank-01.d does have chromatograms in it, and "Blank-03.d" does not have chromatograms.
-- Nick
 
prajita responded:  2022-03-17
Hello Nick,

Thank you so much for you help.

I ran the sample again and it worked now. I have another question:

Now that I have my precursor ions. Can I rank the abundance of these proteins in my sample based on spectral counts or ion abundance?
 
Nick Shulman responded:  2022-03-17
I am not sure that I understand your question.

Skyline does not have any features to help you find out spectral counts. Skyline only knows how to quantify things using chromatogram peak areas.

If you want to learn about how to get lists of data out of your document, including how to sort by many different values, you should take a look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick