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Picking the correct peak failing in ~50% of my injections

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Picking the correct peak failing in ~50% of my injections sophia  2025-07-07 13:04
 

Fairly new to Skyline. I am trying to use it to analyze a large metabolomics dataset. I have RT windows for my analytes set to be pretty tight, like 0.1 min. In roughly half of my injections, Skyline is choosing the wrong peak as the analyte. Screenshots below as an example -- in one, you can see it's picking the peak that agrees with the explicit RT of 4.7 min, and in the other, you can see it's picking a peak nearby at 4.5 min, which has a lower intensity and a higher ppm error, even though the 4.7 min (correct) peak is right there.

Additionally, in other cases, the integration doesn't seem catch all of the peak, or vice versa, not over-integrate outside the peak. It seems like the recognition of where the peak ends is not very good?

I'm having to manually adjust the assignment and integration in individual injections, which is simply not scaleable. I'm not sure why it's performing this way, especially with a tight RT window set, so I'm wondering if there are some parameters in Skyline that I'm unaware of that would make it less error-prone?

Operating system: OS Sequoia 15.5 (24F74)
Skyline version: 64-bit 24.1.0.199

It seems very similar to the issue reported here (https://skyline.ms/announcements/home/support/thread.view?entityId=9a0b237b-a72c-102f-a8bb-da20258202b3&_docid=thread%3A9a0b237b-a72c-102f-a8bb-da20258202b3) -- should this have been resolved in my version of Skyline?
 
 
Nick Shulman responded:  2025-07-07 13:46
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.

The Share Document dialog also gives you the option of including the raw data, which would be helpful in this case.

Files which are less than 50MB can be attached to these support requests.
You can always upload larger files here:
https://skyline.ms/files.url

I am not sure why Skyline would be picking that peak which does not actually overlap with the Explicit Retention Time.
Usually, when people have specified an Explicit Retention Time the complaint is the opposite: Skyline will always choose a detected peak which overlaps with the Explicit Retention Time, no matter how low quality the peak is on other dimensions.

If you want to learn more about why Skyline chose a particular peak, the Candidate Peaks window is useful. You can get to that window with the "View > Live Reports > Candidate Peaks". That window will show you the peaks that Skyline detected and how they scored.

After we see your Skyline document we will be able to give you more information about why Skyline is doing what it is doing.
-- Nick
 
sophia responded:  2025-07-07 13:56
Thank you for taking a look.

I've uploaded the file to linked location, filename: HILIC-W44-1-2_2ndpass.sky.zip
 
Nick Shulman responded:  2025-07-07 14:15
Thank you for uploading that Skyline document.

Can you tell me which molecule and replicate you were looking at in your screenshots?
-- Nick
 
sophia responded:  2025-07-08 05:48
Choline_light

The screenshot with the wrong assignment came from sample I5_pos
An example of the correct assignment is sample H7_pos
 
Nick Shulman responded:  2025-07-08 10:26
I think you might have set the "Explicit Retention Time" after already having imported chromatograms.
If you make a change like that which affects Skyline's opinion of the peaks that it has detected, you often need to use either the "Rescore" or "Reimport" button on the "Edit > Manage Results" dialog.

By the way, when you collect data for each sample in multiple injections like you have in this document where each sample has a "pos" and "neg" file, it works better if you tell Skyline to put the two files in the same Replicate.
When you do "File > Import > Results" the first window asks you how to organize multiple injection files as replicates.
-- Nick
 
sophia responded:  2025-07-08 11:34
Thanks. I re-imported it, but I'm still seeing issues like this one (same analyte as before). I'm not sure why it's picking the other peak when the RT set is closer to the apex of the other, correct peak
 
Nick Shulman responded:  2025-07-08 11:42
Can you send me the file for that Replicate that is "079_Week44-SP1-Plate02_sample_I5_pos.mzML"?
You can upload it here:
https://skyline.ms/files.url
-- Nick