how to remove the peak with 20ppm?

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how to remove the peak with 20ppm? karolina minta  2018-11-01
 

Hello,
I have a problem with removing a peak from Skyline, which has above +20 ppm. In the spectrum I see heavy peptide (-2 ppm) and this light (+20ppm), see attachment. Certainly, the one representing the light peptide is not the peak I am looking for. I tried to decrease ion match tolerance in transition settings ->library and re-import the data, but the peak still appears. Do you know how can I remove it from my spectra?
Thank you,
Karolina

 
 
Nick Shulman responded:  2018-11-01
When Skyline extracts chromatograms from spectra, Skyline adds up the intensities in the scan across a narrow m/z range around the actual m/z of the transition. The width of that m/z channel is determined by the resolution that you have specified on:
Settings > Transition Settings > Full Scan

If you are seeing a high mass error, then it probably means that there is some interference from some other ion in the sample. If you want to reduce the width of the m/z channel that Skyline is summing across, you could change the resolution setting to be a higher number. You can change the mass analyzer to "centroid".

It is likely that you are only getting interference on some of your transitions. If you right-click on your chromatogram graph, and choose "Transitions > Split Graph", then Skyline will display your two precursors in separate panes, and might display each transition chromatogram separately. (If not, then choose something such as "Transitions > Single" or "Transitions > All")

If you click on the chromatogram, you can bring up the Full Scan Graph window, which will show you the spectrum that contributed to that point on the chromatogram. You might see what is interfering there.

I hope this answers your question. I am not sure what you meant about removing the peak.
-- Nick
 
karolina minta responded:  2018-11-02
Hello,

What I meant by removing the peak is that I want Skyline to pick up only the correct peptides and not the incorrect ones.
I assumed that I could set a tolerance for the fragments, say at 10 ppm, and then the +20 ppm peak would not appear.
The +20ppm peak is clearly not the correct one based on the msms spectra (see attachment: y5 is wrongly assigned in picture A, which represents this +20ppm peak (the peak picked is the 3rd isotope), while in picture B y5 is correct).
Clearly it is caused by an interference from another compound in my sample.
 
However, is there any setting I can adjust so that Skyline ignores peaks that are outside the desired mass tolerance?
Changing the resolution from 35000 at m/z200 (which is how the Q Exactive’s operated) to a higher value or centroid doesn’t help (just changes the peaks somewhat); why would that help? It’s the mass deviation that is off.
 
Maybe I don’t understand how the peak picking works.
 
Kind regards,
Karolina
 
Nick Shulman responded:  2018-11-02
You should change the mass analyzer to "Centroided" in the settings on:
Settings > Transition Settings > Full Scan
Also, set the mass accuracy to something small "10ppm".

The peak that you have in the upper half of your "y5" screenshot will collapse down to a single point which will be outside of the m/z channel that Skyline looks at when extracting that particular chromatogram.

After you change settings which affect chromatogram extraction, you need to tell Skyline to reimport the results again, which you can do by going to:
Edit > Manage Results > Reimport

If, instead, you wanted mass accuracy to be one of the characteristics of peaks that Skyline pays attention to when deciding which retention time has the correct peak, you should train an mProphet model. Here's the tutorial for that:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_peak_picking

It might be that the y5 transition is not a good one for you to be including, since the thing in the upper pane of that screenshot has a retention time that is so close to the thing that you are looking at. I cannot say for sure without looking at your raw data.

If you want to send us your Skyline document, we might have better advice.
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 should send us that .zip file, and that .raw file.
You can upload those files here:
https://skyline.ms/files.url

Hope this helps,
-- Nick