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Peak Area Ratio to Heavy Calculation

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Peak Area Ratio to Heavy Calculation erints  2025-03-14 13:58
 

Hello,

I am using Skyline (64-bit) 24.1.0.414 for my analysis. I am trying to compare a few different samples that have heavy labeled peptides to determine the best ratio at which to use our endogenous sample to heavy peptide. I am looking at 2 oxonium ions for 1 heavy and 1 light peptide per sample. I've found the Peak Area - Replicate Comparison bar graph very useful to look at the peak area ratio to heavy, by clicking on "normalized to heavy". This will be very useful as we identify the best ratio and compare normalized samples to our internal standards. I've been using the custom report tool to export each of the heavy and light Total Areas for each sample and peptide. However, which column name do you use in the report tool to project the Peak Area Ratio to Heavy values we see normalized in the graphs? I have attached a file of what my graph looks like, so you can see their approximate values.

I've already tried exporting with Total Area Ratio and Ratio to Standard, but these have completely different values than what my graphs are showing. I've confirmed that the Total Area Ratio is just the Light Total Area / Heavy Total Area, but we would like to know how the Peak Area Ratio to Heavy is calculated and exported, as these are not the calculations we are seeing? The closest I could come up with was the sum of all Heavy and Light Total Area / Heavy Total Area, this gives me a similar curve, but yet the values are still not accurate for all my samples.

For instance: The first sample in the graph has 855524 Light Total Area, 979855 Heavy Total Area, Total Area Ratio of 0.873, however, the Peak Area Ratio to Heavy Graph value is 1.922. The last sample in the graph has 1600330 Light Total Area, 136575 Heavy Total Area, Total Area Ratio of 11.717, and Peak Area Ratio to Heavy Graph value is 25.439.

Thanks for your help!
Erin
 
 
Nick Shulman responded:  2025-03-14 14:32
Can you send me 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

I am not sure I understand exactly what Skyline is plotting in the graph in your screenshot. The Y-axis is "Peak Area Ratio To Heavy" and there are two different colored bars on the graph corresponding to "HexNAc - 204.0866+" and "HexHexNAc - 366.1935+".
The thing that is confusing to me is that the two different colored bars are stacked on top of each other, which is not usually a meaningful thing to do when the thing being plotted is a ratio.
That is, if Molecule A has a light:heavy ratio of 0.1, and Molecule B has a light:heavy ratio of 0.2, the number "0.3" does not really have any physical significance even though, technically, it is the sum of Molecule A's and Molecule B's light:heavy ratios.

For this reason, Skyline is supposed to display a different sort of graph when you are plotting more than one molecule like that. Either it is supposed to be a line graph or the different colored bars are supposed to be next to each other. Anything so that it won't look like the values are being added together.

In addition to sending your Skyline document it would also be helpful if you could include a screenshot of the entire Skyline window so that I can see what you have selected.

The "Total Area Ratio" and "RatioLightToHeavy" are the columns in the Document Grid that you could export to get the ratio of your light and heavy peak areas.
Additionally, if you go to the Quantification tab at "Settings > Molecule Settings" and set "Normalization Method" to "Ratio to Heavy" then the column "Normalized Area" in the Document Grid will also tell you the light:heavy ratio for each Molecule result.
You can use the binoculars button at the top of the Report Editor to search for any column by name.
-- Nick
 
erints responded:  2025-03-17 11:42
Hello,

Attached is the skyline file with the chromatogram results. I realized when I click on Normalize to "Default (Heavy)" the bar graph shows the stacked sum peak area ratio to heavy, but when I click on Normalize to "Heavy" the two oxonium ions are split side-to-side as you described.

I also included a file with a screenshot of what I clicked on, as requested, which was my light peptide for one of the glycopeptides. In this file, I also included a few screenshots of reports that included the "Total Area Ratio", "RatioLightToHeavy", "Ratio To Standard" and "Normalized Area". These all contained the same values as Total Area Light / Total Area Heavy.

However, I still can't find what report columns reproduce the values I'm seeing in the "Peak Area Ratio to Heavy" graphs. I added screenshots of what these values are as I hover over the first bar in the graph. Maybe you are able to find out how these are calculated and why they're not the same as the columns I've already added to my reports?

We are open to both the Total area ratio as a sum of the oxonium ions and analyzed separately as you suggested, however in my reports, these ratios are always the same even when they are separated via their respective oxonium ions as if they were summed together. You can see this in the screenshot file as well. Am I not looking at this in the right view to see these differences, as I would expect them to be different?

Thank you,
Erin