one point quantification dms73  2021-08-05
 

Hi -

I plan to run a verification study of label free results and will be targeting 50 peptides. I have heavy labelled peptides that I plan to include in each sample. I would like to quantify with Skyline using a signal point curve. Does Syline have this quantification option?

Thanks,

Danie Schlatzer

 
 
Nick Shulman responded:  2021-08-05
What exactly do you mean by "one point quantification"?

We use the term "single point calibration" when there is an external standard (i.e. a separate well on the plate) which contains a sample with known concentrations. Skyline can do quantification relative to that external standard. You would do this the same way as you would multi point calibration where you use the Document Grid to set the "Sample Type" of that replicate to "Standard", and you would set the Analyte Concentration of that replicate to a number. Usually you would set the Analyte Concentration of the external standard to 1, and then you would set the Concentration Multiplier of each of the peptides to the actual concentrations of the peptides.
Then, you would go to "Settings > Peptide Settings > Quantification" and set the "Regression fit" to "Linear through zero".
After you do all that, the "Calculated Concentration" column in the Document Grid would tell you the concentration of each peptide in each replicate.

Here is the absolute quantification tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_absolute_quant

It sounds like you might be asking about how to do internal standard quantification, where you do not have an external standard, but you would like Skyline to multiply the observed light:heavy ratio by the concentration that you know you spiked your internal standard in at. Skyline can do that too. To do internal standard calibration, you should use the Document Grid to set the "Internal Standard Concentration" of each Peptide to the appropriate number.

If you would like to learn more about the Document Grid you should take a look at the Custom Reports tutorial:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

Hope this helps,
-- Nick