I am not sure I understand your questions.

When you have multiple external standards, all of those points get plotted on the graph, and Skyline does a linear regression through all of the points.
Why would you want to average the points before doing the linear regression? I understand the concept of averaging points when you have technical replicates, because you don't want the multiple points to get too much weighting, but I cannot think of a reason that you would want to do this with a calibration curve.

By the way, this is our tutorial for calibration curves in Skyline:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_absolute_quant

I also do not understand your question about Leucine and Isoleucine.

This tutorial about iRT retention time prediction might be helpful to you:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_irt

-- Nick

Thank you for your quick response and the links. We wish to do the averaging of the XIC areas first before the linear regression. Exactly like you said, right now it's using all the points for calibration curve plotting and for unknowns it's using each replicate to quantify. We'd like to get the average for each standard level and treat it as single value when plotting the standard curve. This is more commonly used in plotting standard curves when each measurement is not independent. We're doing the replicates in the same experiments and the points we get for each is not independent. For more on whether to use the average for standard curve, please see this article. https://www.graphpad.com/support/faq/if-i-give-prism-replicate-values-as-opposed-to-the-means-of-those-values-will-the-curve-fit-parameters-reported-be-the-same-which-approach-is-better/

For the Leu and Iso-Leu, right now we're getting the sum of the XIC peak areas for both amino acids and we hope to get the peak area for each separately so that we can quantify them separately. Hope this has clarified the questions.

Are you saying that when you did your calibration curve, the mass spectrometer sampled the same well on the plate multiple times for a given concentration level, rather than having multiple wells on the plate that had the same concentration level?
In that case, I guess it would be appropriate to average the points before doing the regression.

When you are doing a group comparison to calculate fold changes between cohorts, Skyline does allow you to specify a "BioReplicate" annotation which causes things to get averaged before doing the linear regression. Here's the tutorial for group comparisons:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_grouped

It might be that we should extend that BioReplicate stuff so that you can specify it for calibration curves. We will try to think about this.

There are many ways to tell Skyline what you expect the retention time of a peptide to be. One way is by specifying the "Explicit Retention Time" for the peptides in the Document Grid. Here is the tutorial that teaches about the Document Grid:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=tutorial_custom_reports

-- Nick

Thank you Nick! It's very helpful info.