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accuracy of light and heavy precursors differing by 1 Da only

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accuracy of light and heavy precursors differing by 1 Da only rameshkr  2025-02-28 04:02
 

Hi team

I have two peptides light and heavy (8 amino acids long). The heavy peptide has a single 13C on the 3rd amino acid so differs from light peptide by 1 Da only. I want to spike the heavy peptide in the light and monitor their +2 charge precursors (m/z difference 0.5 only). I have generated a skyline document and it gives me details for both but I was wondering how to verify that the peaks are being correctly identified- I am getting same product ions for both. Also, the M+1 and M+2 of light is same (upto 3 decimal places) as M and M+1 of heavy. I am acquiring data on Thermo Q Exactive Plus in PRM mode using 1.6 m/z isolation window and 0.5 m/z offset. I have set skyline ion and method match tolerance to 0.01 m/z under the transition settings.

I have shared the skyline document and raw files on the portal (file name: light and heavy mix_1-13C). It has 3 files, standard- mix of purified light and heavy peptide and biological- two technical replicates of heavy peptide spiked in a tissue peptide extract. The results of all three are confusing.
In the standard, the M of light peptide hasn't been identified but skyline has identified light specific transitions. Can it be correct? Additional replicate give same result.
In biological, precursors and products both are identified for light peptide and only precursor for heavy peptide. If precursor has been identified, why not transitions?
In biological 2, both precursors and product ions are identified for both peptides. Additional replicates give this result. But are the identifications correct?

My aim is to normalize peak intensity/areas of light with heavy. How should I do that?

Thanks
 
 
Nick Shulman responded:  2025-02-28 17:00
Unfortunately, Skyline does not have the feature "deconvolute overlapping isotope distributions" which would accurately tell you how much of the light and heavy peptide you have based on the MS1 peak areas.
All of the peak area numbers that you can see in Skyline make it look like both the light and heavy peptides are both present.
However, if you look at the "Isotope Dot Product" values, you can see that the dot products for the light peptides are very low (close to 0.5) and the dot products for the heavy peptides are much closer to 1 which probably means that the signal that you are seeing is 100% coming from the heavy peptide.

If you want to know the amount of light and heavy peptide that is present, there is enough information to calculate that in the attached CSV file "IsotopeDistProportionArea.csv".
That CSV file has the column "IsotopeDistProportion" which is the expected relative signal for a particular m/z, and the "Area" which is the observed amount of signal.

There is some matrix math that you can do with a least squares fitting to determine how much of the observed signal is probably coming from the light and heavy peptides.
The first part of this poster talks about how to do this for MS1 data:
https://skyline.ms/announcements/home/support/download.view?entityId=f194e395-aeb5-1036-8cc6-e465a393e3d4&name=NickShulmanAsms2018.pdf
(the second part of that poster talks about how it's possible but probably too difficult to do the same thing for MS2 data)

I have been supposed to implement this algorithm in Skyline for a long time.
Let me get back to you in a few days. I might be able to either add this as a feature to Skyline-daily, or I might be able to find you another solution that you can use that will work with data that you are already able to export from Skyline.
-- Nick
 
rameshkr responded:  2025-03-17 21:22
Hi Nick 

Thank you for your response. Have you been able to find something for this?

The standard mix contained the light and heavy peptides in equimolar ratio. The csv file you shared, it shows 0 area for light precursor 523.77 even in standard mix. This can't be true. 
Also, there is no signal for y7. This is the product whose m/z differs between the light and heavy. Can you suggest some ways to get this one?

And I am unable to open the skyr file you shared. 

Thanks a lot for your support
Ramesh
 
Nick Shulman responded:  2025-03-17 21:51
The chromatogram peak area of the monoisotopic light precursor m/z (523.7745++) really is zero.
Here is a screenshot of what those precursor chromatograms look like.
The monoisotopic light precursor is shown in blue in the upper panel, and it coincides with the X-axis between the peak integration boundaries.
Either there is no light peptide present in the sample or those are the wrong integration boundaries (i.e. the peptide has a completely different retention time and we are looking at the chromatogram for a different molecule).

By the way, the way to read a report definition from a .skyr file is to go to "File > Export > Report" and then press "Edit List" and then "Import".
(there is another way to read in report definitions involving the "Manage Results" menu item in the Document Grid).

You can learn more about custom reports and the Document Grid in Skyline here:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports
-- Nick