Hi Bin,
Can you upload a ZIP file? containing your Skyline document (use File > Share to create a .sky.zip) and one of the files that works and one that does not to:

http://skyline.ms/files.url

Then we can have a look and let you know more about what is going on.

--Brendan

Thank you for the quick response!

I uploaded a complete skyline file and 2 Quantiva raw files.

Please let me know if you need anything else.

Thank you very much!

bin

Thank you for sending us your data files.

The problem is that Skyline is not finding your iRT standard peptides at the correct time in the chromatogram. Because of that, when Skyline tries to do a linear regression with your iRT standards, the correlation is too bad, and Skyline fails to calculate the slope and the intercept.

When that happens, Skyline does not import the chromatograms for the rest of your peptides.

I thought Skyline was supposed to give you an error message when that happened.

With your dataset, you are only monitoring two transitions for each of your iRT peptides. It looks like that is not enough for Skyline to be able to reliably find your iRT peptides.

I would have thought that you would be able to work around this problem by editing your iRT calculator and unchecking the "Auto Calculate Regression" box and providing your own slope and intercept. However, even when I do that, Skyline still refuses to import the rest of the peptides if your iRT peptides do not lie on a straight enough line.

I hope this makes sense.
We definitely need to make it so that Skyline gives you an error message when this happens.

Thank you so much! It makes sense. I will put in more transitions for iRT standards in the future.

bin