It sounds like you are trying to tell Skyline about some peptide sequences which contain non-standard amino acids such as "AIB" (2-Aminoisobutyric acid).

I believe the way to do this in Skyline is to specify a peptide sequence consisting of some of the 20 standard amino acids, and then apply modifications to some of the amino acids so that their chemical formula ends up being what you want them to be.

You should not use the letter "X" in your peptide sequence. When Skyline sees a "X" in the peptide sequence, Skyline treats it as an amino acid whose mass is 111.06. I believe that number has something to do with the hypothetical amino acid "Averagine".

If you want to put something in the amino acid sequence that has a mass of zero, the letter to use in Skyline is "J".

Then, you would go to:
Settings > Peptide Settings > Modifications
and push the upper "Edit List" button to define modifications whose chemical formula can transform standard amino acids to the non-standard amino acids that you need.
You can right-click on the peptide sequences in the Targets tree and choose "Modify" to apply these modifications to the correct amino acids.

I have never seen a Skyline document where anyone was trying to analyze nonstandard amino acids, but I would expect this to work.
-- Nick

Alternatively, you could use J and then make a J-specific modification with the chemical formula for the entire non-standard AA. This would save you having to do math to calculate atom delta between your AA and some other standard AA.

I just tried this with "2-Aminoisobutyric acid" where Wikipedia gave the chemical formula:

https://en.wikipedia.org/wiki/2-Aminoisobutyric_acid

I ran into the fact that Skyline only offers the 20 standard amino acids for modification. So, I had to leave it non-specific modification and then add it to J using Edit > Modify Peptide, but otherwise, it seemed to work pretty well to me.

PEPJ[+103.1]IDER
    479.7456++
        P [y6] - 732.3886+
        J [y5] - 635.3359+
        I [y4] - 532.2726+

I think we should consider allowing modification definitions specific to J, since this seems pretty useful to me for at least including 1 arbirarily defined non-natural amino acid in a document.

Interesting post. Thanks for sharing your case on the Skyline support board.

--Brendan