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:
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: