Thank you for sending us your files.
Skyline makes two passes through your .wiff file to extract chromatograms. During the first pass, Skyline only extracts chromatograms for your iRT peptides. Then, Skyline does peak picking on those iRT peptides, and then attempts to do a linear regression between the iRT values and where the peaks were found, in order to find the correct slope and intercept to use for mapping between iRT values and retention times.
Unfortunately, the places where Skyline finds your iRT peptides is not consistent with their scores, and so the R-squared of the linear regression ends up being too small, and so Skyline does not proceed to the second phase of extracting chromatograms for the rest of your peptides.
Skyline is supposed to give you an error message when this happens, but there seems to be some sort of bug that prevents this error message from being shown to you. I will try to figure this out.
You might be able to look at the chromatograms for your iRT peptides and figure out why Skyline is not able to find them where they are supposed to be.
It might also be helpful to look at the graph:
View > Retention Times > Regression > Score To Run
in that Retention Time Regression view, make sure you right-click and choose "Calculator > LPS_IMAC" and "Points > Standards" and "Replicate > Single" in order to see just the iRT peptides for the replicate that you are interested in.
-- Nick |