Using SIS-only calibration curve to quantify light or light/SIS ratio david schmidt  2021-03-18
 

Hi,

I'd like to use a calibration curve generated with heavy (SIS) peptides only (no light peptides present) for the quantification of either light peptides or light/heavy ratios (SIS peptides as internal standards). Is that possible?

Thank you!

David

 
 
Nick Shulman responded:  2021-03-18
Are you asking about reverse calibration curves? Your question might be answered by Brendan's answer in this support request:
https://skyline.ms/announcements/home/support/thread.view?rowId=36322

The reverse calibration curve cannot be used by Skyline to calculate the concentration of unknown samples, but you might be able to use it to determine the linear range of your instrument.

Then, when you spike your internal standard into your unknown samples, you can use the Document Grid to set the "Internal Standard Concentration" on each peptide.
When the "Internal Standard Concentration" is set, Skyline will multiply the light:heavy ratio by that value, and that product will be available in the Document Grid in the "Calculated Concentration" column ("Peptide Results > Quantification > Calculated Concentration").

Hope this helps, and let me know if you were asking about something else.
-- Nick
 
david schmidt responded:  2021-03-18
Hi Nick,

thanks for the swift response. No, I didn't mean a reverse calibration but rather using only the SIS version of each peptide for calibration. So one would simply get Intensity vs. concentration for the SIS peptide and then apply this to the light versions in each sample. Since it should behave identical both chromatographically as well as in MRM, that should be a valid approach.

So what I'd like to know is: Once I generated a calibration curve for each SIS peptide (Peptide settings -> Internal Standard Type = none; Normalization Method = none), can I somehow transfer the slope+intercept and use it to quantify the light versions in samples? If not in Skyline, do you happen to know whether this is possible in Quasar or MS Stats?

best, David
 
Nick Shulman responded:  2021-03-18
Skyline does not have the ability to use the other slope and intercept to calculate the concentration of your peptides like that.

If all that you cared about was the slope, you could achieve this by using the Document Grid to set the "Internal Standard Concentration" for the peptide. Skyline will multiply the Internal Standard Concentration by the ratio to heavy in order to get the calculated concentration.

I could imagine that you might be able to achieve what you want in Microsoft Excel. You can use the Document Grid to export the slope and intercept of the calibration curve.

We have gotten several requests to be able to use other molecules' calibration curves. One example is here:
https://skyline.ms/announcements/home/support/thread.view?rowId=49859

We might be able to implement this in a future version of Skyline. It might be helpful if you could send us your Skyline document and so we can make sure that the future feature will solve your needs.

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:
https://skyline.ms/files.url

-- Nick