problems about quantity small molecular compounds

problems about quantity small molecular compounds kaylee xu  2019-07-22


I have a question about skyline when I want to quantity the small molecular compound.

I want to get the concentration of SM d18_1-12_0 use the calibration curve of 18:1(d9)SM (Please refer to the attachment). Is it possiable?

Thank you very much!

Nick Shulman responded:  2019-07-22
I am not sure that I understand your question.
The calibration curve for SM d18_1-12_0 is completely wrong. There is no correlation between the specified concentration of the samples and the observed peak area. This probably means that you are looking at the peak area of something which is not the compound that you spiked into your samples.

It might help if you sent 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.
Otherwise, you can upload it here:
-- Nick
Brendan MacLean responded:  2019-07-23
Agreed. It looks like the peak area is remarkably stable (around 2,000,000) across 5 samples. Are you saying you want to estimate the concentration for a peak area of 2,000,000 from the calibration curve for 18:1 (d9) SM? That is not usually how we do things, but ... interesting that the calibration curve graph pane does not list an intercept but only a slope. But, that slope is 795,000. So, if you know the concentration of 18:1(d9) SM for a peak area of 1,765,961 is 2.22 (not sure about units), then you can roughly estimate the concentration of SM d18_1-12_0 for a peak area of 2,065,800 as 2.22 + 300,000/795,000 = 2.6.

I guess I would need the full regression line equation for 18:1 (d9) SM to do a fully correct transferral of that equation to the peak area of SM d18_1-12_0. I can't really say whether doing that is valid, but is that what you are looking for? I think you can get both the slope and intercept for the curve from the Document Grid by adding the Calibration Curve column to a report. The you just do the simple equation Calculated Concentration = Peak Area * Slope + Intercept. Is that what you are trying to do? I was expecting to be able to do that myself from the text notes on the Calibration Curve graph you provided, but can't see an Intercept value.

We don't have an automatic way to assign a calibration curve for one analyte to the peak area for another. Maybe you can explain a bit more why that makes sense in your case. It is the first time we have heard a request for that.

Thanks for the screenshots.

kaylee xu responded:  2019-07-23
In Lipidomics research, it is a common way to calculate the concentration of lipid species with its deuterated isotope labeled analytes. So can you help me to calculate the concentration of SM d18_1-12_0 or else with the equation to the peak area of 18:1 (d9) SM?
Brendan MacLean responded:  2019-07-23
It sounds like you are saying that SM d18_1-12_0 is the same molecule as 18:1 (d9) SM with some (or all) H atoms deuterium-labeled (i.e. 2H).

Skyline expects this to be expressed by having a single "molecule" target with multiple "precursors" differentiated by their adducts. I will let others chime in on how to set this up.

I am also not quite positive that Skyline support multi-point calibration of the light concentration from a heavy response curve, like your data. I believe this is also somewhat common in proteomics, but not the way we and our closest collaborators do this. We will have to discuss more how we might support this better.

Thanks for your document. It clarifies why Skyline doesn't show a y-intercept for the calibration curve linear equation. It is because you used "Linear through zero" for your Regression fit in the Molecule Settings > Quantification tab. That means the y-intercept is zero. So, to get a concentration from a peak area, you would just divide the peak area by the slope. i.e. concentration = peak area / slope. That is your equation.

Or in the original example:
2,065,800/795,000 = 2.6 (same result as before)

Hope this helps. We will continue to consider what we might do to support having calculated concentrations in the Document Grid for this method of quantification.

Thanks for the information you have provided.

Brian Pratt responded:  2019-07-23
As Brendan says, Skyline will understand the labeling relationship if you have single molecule with multiple adduct descriptions - one for the unlabeled variety, and one for the labeled.

For example, here's a transition list with a single molecule with a light and heavy (D5) precursor

Molecule List Name,Molecule,Precursor Formula,Precursor Adduct
My List,My Mol,C8H10N4O2,[M+H]
My List,My Mol,C8H10N4O2,[M5D+H]

Pasting that into the Targets window results in a Document Grid "Precursors" report that looks like this:

Precursor,Molecule,Precursor Charge,Isotope Label Type,Precursor Ion Name,Precursor Ion Formula,Precursor Neutral Formula,Precursor Adduct,Precursor Mz
195.0877[M+H],My Mol,1,light,My Mol,C8H11N4O2,C8H10N4O2,[M+H],195.087652
200.1190[M5D+H] (heavy),My Mol,1,heavy,My Mol,C8H6H'5N4O2,C8H5H'5N4O2,[M5D+H],200.119035


Brian Pratt