Chromatogram unavailable issue

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Chromatogram unavailable issue Bo An  2019-04-15
 

Hi everyone,

I encountered a "chromatogram unavailable issue" with Sciex MS2 data, I have two transitions for the targeted peptide, if I only select one transition in Skyline method, the chrom is ok for both; but if I included both transitions in the Skyline method, only one chromatogram will be displayed, the other one will not be recognized, have any body encountered this issue before?

Many thanks,
Bo

 
 
Nick Shulman responded:  2019-04-15
Bo,

If you add transitions to your Skyline document, you need to tell Skyline to extract the chromatograms again in order to see MS2 chromatograms for the new transitions. You can do that with the command:
Edit > Manage Results > Reimport
This is what you do for chromatograms which were extracted from full scan MS2 data.

If instead you have SRM data, then it usually is not necessary to do a reimport after adding transitions, since Skyline usually copies all of the available SRM chromatograms into your .skyd file. With SRM data, missing chromatograms are usually caused by there not being any matching SRM chromatogram in the raw (or .wiff) file. Sometimes with SRM data it is necessary to set the "Method match tolerance m/z" to a higher number. That number is at:
Settings > Transition Settings > Instrument

If neither of these things fix your problem, then you should send us your data.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including extracted chromatograms.
You can send us that .zip and one of your .wiff (and .wiff.scan) files.

If those files are less than 50MB, you can attach them to this support request.
Otherwise, you can upload them here:
https://skyline.ms/files.url

(but, you probably just need to do a "reimport").
-- Nick
 
Bo An responded:  2019-04-16
Hi Nick,

Thanks a lot for your response. I did the steps you suggested. Please find the uploaded file as you suggested. The name is POST_AAA.sky.zip. You will find two product ions for the peptide. If I un-select one product ion, the chromatogram for the the selected one will show up, but if I select both, only one of the chromatogram will show up. I guess data for both chromatogram is there, but they can not show up simultaneously. Thanks. Any suggestion will be appreciated.

Best,
Bo An
 
Nick Shulman responded:  2019-04-16
Hi, Bo,

I see that this is an SRM experiment. The problem is that the Q1 values for each of your transitions is slightly different.
That is, you have two chromatograms which are both supposed to belong to the same peptide:
Q1: 700.89 Q3: 1074.58
Q1: 700.891 Q3: 973.53

Because of that .001 difference in the Q1 value, Skyline assumes that those two chromatograms belong to different peptides.
You have a peptide with the precursor m/z 700.8908 and product m/z's 1074.5790 and 973.5313.
In this case, Skyline is either going to show you the chromatogram with Q1 700.89 or Q1 700.891, never both.

The best thing for you to do is collect this data again, and make sure that the Q1 values that are for the same molecule match exactly.

You can tell Skyline how closely Q1 and Q3 values have to be to match the calculated m/z of precursors and transitions. That setting is called "Method match tolerance m/z" and it is on "Settings > Transition Settings > Instrument". This setting does not affect the grouping of SRM chromatograms. For SRM chromatograms to get grouped together, their Q1 values have to match each other exactly.
-- Nick
 
Bo An responded:  2019-04-17
Hi Nick,

Got it, thanks a lot for the information. Problem solved.

Best,
Bo An