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Defining retention time for peptide quantification

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Defining retention time for peptide quantification mt  2023-03-28 02:20
 

Hi, I started using Skyline (version 22.2.0.351) recently and have been trying to set up a peptide peak quantification protocol but I have trouble effectively defining retention time windows for peak detection.
I already have individual retention times for peptides of interest and would like to narrow down the window in which skyline searches for them. Very similar to this support case.

I managed to define the Explicit RT and Explicit RT Window in the Document Grid, but the issue I keep having is that the right peak is picked only if Explicit retention time falls exactly within a peak. More precisely, it has to fall within a peak integration boundaries that Skyline does defines every time, but are presented as either black or grey dashed lines, depending on the picking success. If the defined explicit retention time is outside of these boundaries, random noise is picked near the defined retention time, even though the actual peak might be eluting just a few seconds before or after the explicit RT and is well within the RT window. Re-scoring and re-importing doesn't help.
Is this behaviour expected and and is there a way to make skyline pick the actual best peak within the defined window? It could be that the problem is related to ppm which is sometimes lower for the picked noise than the actual peak. I have attached a screenshot of the chromatogram where the issue is visible.

Thank you!
 
 
Brian Pratt responded:  2023-03-28 14:43

Explicit RT is really only appropriate for stable chromatography - it ignores anything outside the specified window by design. If you have a peptide that doesn't elute consistently you'll need to give it a wider window.

Thanks for using the Skyline support board,

Brian Pratt
 
mt responded:  2023-03-29 00:32

Hi Brian,

thank you for the answer. I understand that anything outside the RT window will get ignored. My issue is that peptides seem to get ignored also within the window. Shifts in retention time I experience are in seconds while my RT window is 1.5 - 2 minutes, so it always covers the whole peak. On the screenshot I provided you can see the RT window in yellow and the peptide peak well within it, but skyline picked something from the noise right next to it, more or less exactly at the Explicit RT. In other replicates where the peak elutes within this time point it will get picked properly. Maybe I am not fully understanding how this should work, but my expectation was that skyline will try to find the best peak within the whole window and not just at the RT point around which the window is defined. Is there maybe a way to specify the minimum signal intensity or some additional parameters that would help the right peak to be picked?

Thank you again
 
Nick Shulman responded:  2023-03-29 09:19
Can you send us your Skyline document?

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 is less than 50MB you can attach it to this support request.
You can upload larger files here:
https://skyline.ms/files.url

Whenever Skyline seems to be picking the wrong peak, I recommend using the menu item:
View > Other Grids > Candidate Peaks

The Candidate Peaks grid shows you the list of peaks that Skyline detected and how each of the peaks scored.
In your case, I think what is happening is that when a peak happens to overlap with the "Explicit Retention Time" that you have specified, Skyline ends up giving that particular peak a really good score in the "Identified count weighted feature" column.

We might be able to come up with a solution after we see your dataset.
-- Nick
 
mt responded:  2023-03-30 07:01

Hi Nick,

Thanks a lot for the answer! I have uploaded a simplified version of our data set as "Explicit RT support case.sky.zip" where the issue is present for some samples (e.g. Sample10 for K.DLGEEHFK.G peptide).
I checked the very revealing Candidate Peaks grid and you are right, "Identified count weighted feature" column dominates the score even though almost all other parameters are better for the correct peak (Screenshot_Sample10). I found this tutorial where the parameter is described as "number of identified peptides within the peak boundaries" even though I still don’t understand why the correct peak has zero. After quickly editing the Peak scoring model by disabling "Identified count" feature, the results seem to have improved. Is there a better way to approach this?

Also, I found some replicates where the correct peak was not even integrated (Screenshot_Sample9), and therefore cannot be picked. I assume this is a separate issue, but if you have some suggestion, that would also be great.

Best,
Marko
 
Nick Shulman responded:  2023-03-30 14:42
Thank you for uploading that Skyline document.
We will try to figure out a way to make it so that Skyline does a better job in this scenario.

By the way, if you want to integrate a time range where Skyline has not even found a candidate peak, you can click below the X-axis and drag horizontally.
The Existing Experiments Tutorial reveals this technique for choosing peak boundaries on page 15.
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_existing_quant
-- Nick
 
mt responded:  2023-04-04 03:46
That’s great, thank you for your help and suggestions!
Best,
Marko