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Peak Integration: PRM owagne2  2025-04-29 13:42
 

Hello,
I spiked in my heavy peptide into the sample matrix (cell digest) containing my endogenous peptide, but during the analysis on Skyline I noticed that the endogenous peak appeared to have split into multiple peaks. When I was looking through other requests about peak splitting, Skyline staff members previously warned against integrating differing retention times between the heavy and endogenous (light) peptides, which of course makes sense.

So, my question is this: How do I handle this peak integration, if at all? From where I'm sitting, the endogenous peak doesn't appear to be quantitative, but I'm not familiar enough with Skyline to figure out if the problem is a lack of knowledge on my end, a sample degradation problem, a meaningful biological artifact, or a methods/sample preparation problem. For reference, I am running a PRM proteomics experiment on Skyline version 24.1.0.414.

I've attached images of the heavy peak, the endogenous peak that Skyline recommended I integrate, the endogenous peak that I manually wondered if I should integrate, and an overlap of the precursor ions for both the heavy and light peptides (blue is the heavy, red is the endogenous).

I first assumed that I have a PTM on my endogenous peptide, but that would change the overall mass and my PRM scan wouldn't have picked up my peak. I have an amino acid in my sequence that perhaps could have undergone racemization (my cell pellets were >1 yr old), but I wasn't sure if there was a simpler or more likely explanation before I attempt a new experiment. For reference, this peptide comes from a membrane bound glycoprotein. To my knowledge, there are no known glycosylation or phosphorylation sites on my peptide.

I apologize for the novel, but I thank you for your time and look forward to your response.

Sincerely,
Olivia
 
 
Nick Shulman responded:  2025-04-29 14:24
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.

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

Which particular peptides are you seeing these double-elutions with?
Peptides containing Proline often have two distinct retention times. The reason for this is that Proline is a big amino acid, and the two ends of the peptide might in different conformations around that big amino acid, resulting in different retention times for the same type of molecule depending on the exact shape that it has at the time.
For the Proline-containing peptides that have two distinct retention times, it is recommended that you choose a different peptide for quantification because it is too difficult to try to get a meaningful quantitative number from those two different chromatogram peaks.

There are other times when you will see very similar chromatographic peaks near each other that actually come from different molecules. For instance, some amino acids lose one Dalton in mass when they are deamidated, and those deamidated peptides have many fragment ions in common with the original peptide, and sometimes the deamidated chromatogram peak is chosen instead of the correct one. For deamidated peptides, it is important to make sure you are indicating the peak with the good MS1 Isotope Dot Product (idotp).

Let us know which peptides you are interested in. After we see your Skyline document we will probably be able to give you better advice.
-- Nick
 
owagne2 responded:  2025-04-29 15:57
Hi Nick!

I uploaded my Skyline document ("Troubleshooting") to the box folder, as it was rather large. I'm still very new to Skyline, so it is quite possible that I have an incorrect setting or display.

I don't have any prolines in the peptide I originally messaged about, but I do have prolines in some of my other peptides. I'll certainly keep that in mind for the future.

I apologize, would you be able to tell me how to display the idotp?

At present, I'm interested in the "LQGET..." peptide, as it had the best signal and dotp.
 
Nick Shulman responded:  2025-04-29 16:34
The Isotope Dot Product can only be calculated from MS1 chromatograms.

If you have acquired MS1 spectra you could tell Skyline to extract MS1 chromatograms by changing the settings on the Full Scan tab in Transition Settings.

The MS1 chromatograms are a good way to verify that the molecule you are looking at has the correct precursor m/z.

In this case, it is fairly obvious that the large peak to the left of the peak that Skyline chose is not the correct molecule. The ratios of the transition peak areas do not match the library spectrum. That is, the library dot product (dotp) is not a good value.
--Nick
 
owagne2 responded:  2025-04-30 09:26
Thank you for your help! That all makes sense.