When you have a multi-injection replicate, I find it nearly impossible to use the Retention Time regression graph to figure out what has gone wrong with the iRT regression. If you are trying to figure out why Skyline was not able to do the iRT regression for a particular .raw file, you might want to import that .raw file by itself into a separate replicate. The Retention Time Regression graph shows you the measured retention time of your iRT standards averaged across all of the injections, which makes it hard to figure out what went wrong if a particular peptide was found at the wrong time in only one of the injections.

Also, when you are trying to figure out this insufficient correlation error, you should tell Skyline to only show the iRT standards on the graph. That is, on the right-click menu of the Retention Times Regression window, you should choose the menu item "Points > Standards". I believe all of the purple dots towards the left edge of your retention time linear regression graph are the peptides which are not in your iRT database. Their presence on the graph makes it really hard for you to figure out which of your iRT standard peptides are being found at the wrong measured retention time. Those dots will probably disappear if you choose "Points > Standards".

If you send us your files, I can try to help you figure out what is going wrong.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files including iRT database.

You should also send us your .raw files.

You can upload those files here:
https://skyline.ms/files.url

I've attached the document and the numerous data files.

Individual replicate still gives insufficient correlation error.

Thank you for those files.
The "Apomyoglobin_APH_All_01NOV_Test_Last.sky.zip" file does not actually have your iRT calculator set up in it. You must have been playing with different settings and set the Retention Time Predictor to "None".

Can you send me a .sky.zip with your iRT calculator set up in it?

By the way, I think you should have told Skyline to put each of your .wiff files into separate replicates. That is, it looks like "Sample1.wiff" contains 4 injections from the same test tube, and "Sample2.wiff" contains 4 injections from a different test tube?
By default if you were to import results from Sample1.wiff, Sample2.wiff, and Sample3.wiff, Skyline would have given you 12 replicates.
On the "Import Results" dialog, Skyline gives you options "Add single injection replicates in files", "Add multi-injection replicates in directories", "Add one new replicate", and "Add files to an existing replicate".
There's no option for "Add multi-injection replicates from multi-sample wiff files", so I think what you need to do is import the .wiff files one at a time choosing "Add one new replicate" and change the name of the replicate for each of your .wiff files.

Hi Nick,

I'd like to thank you very much for your assistance. I changed the import results and have the apo-std calculator as the default.

Thank you for sending me that .sky.zip.
It looks like your iRT standard peptides have perfect correlation. The only thing that Skyline does not like is that you have only 7 peptides, and Skyline requires at least 8.

One thing that you can do is to add another peptide to your set of iRT standards. Leucine and Isoleucine have the same mass, so you could just add the peptide "ALEIFR" which will behave the same as your existing peptide "ALELFR". (see attached .sky.zip file).

I am not sure exactly why Skyline is imposing this 8 peptide minimum here. I suspect that we have a bug in our logic in this particular spot, and the minimum right here is supposed to the lesser of either 8 or the number of iRT standards, whichever is smaller.
This seems to be only an issue when adding measured results in the iRT calculator dialog. When you were importing results, Skyline had no problem doing the regression with only 7 peptides.

I will look into this and probably it will be fixed in the next update of Skyline-Daily.

Hi Nick,

That did it. Thank you for all your help. Could not have done it with you.

All the best,

Johnny