Non-Canonical Amino Acid

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Non-Canonical Amino Acid lulmer  2022-10-24
 
Hello,

I have a unique use case working with the non-canonical amino acid, benzoylphenelaylanine, and I'm having trouble importing my results into skyline. I'm trying to import comet/peptideprophet search results that have the benzoylphenelaylanine non-canonical amino acid defined (attached interact.pep.xml). When I import these results there is a suggested modification that makes the peptide with the non-canonical amino acid viewable in skyline (attached jpegs 1 and 2 described the modification). However, when I try to reopen the skyline document with that modification I get the error "Variable modifications must specify amino acid or terminus" (attached error.jpeg). I've attached the skyline document that gives this error as well. Any suggestions on how to make these comet/peptideprophet search results with the non-canonical amino acid compatible with skyline would be greatly appreciated.

Thanks!
Lindsey Ulmer
 
 
Nick Shulman responded:  2022-10-24
Attached is a fixed version of "T_noUV_dim_BPA.sky". You should be able to save it overtop of the file of that name which you already have (so that it ends up in the same folder as "T_noUV_dim_BPA.skyd") and then open it without error in Skyline.

If your peptide search results have obscure modifications, we usually recommend that you go to:
Settings > Peptide Settings > Modifications
and define the structural modification exactly as you would like it to be with the correct chemical formula and amino acids that it applies to.

After you do that, you can do "File > Import > Peptide Search" and Skyline will not need to ask you about the modifications that it finds in the .pep.xml file.

Instead, what it seems happened is that Skyline happened to find a unimod modification which was the right mass.

It sounds like there is a bug in Skyline which allowed a modification to be marked as variable which was not specific to any particular amino acid or terminus. Skyline is not supposed to allow that to happen, but I imagine there's a bug where if the modification gets added in the Import Peptide Search wizard it does the wrong thing.

By the way, if you would like to learn more about modifications in Skyline, in particular explicit and variable modifications, this is a good webinar to look at:
http://skyline.ms/webinar10.url

-- Nick
 
lulmer responded:  2022-10-24
With the updated file, I get the error of "Invalid amino acid 'B.'" I also find that I cannot type B as an amino acid under the Settings > Peptide Settings > Modifications window.

Thanks!
Lindsey
 
Nick Shulman responded:  2022-10-24
Oops. My mistake. Try the attached file now.
-- Nick
 
Nick Shulman responded:  2022-10-24
I think other things are not going to work for you, since you are using "B" as the amino acid, and your peptide search engine's idea of what "B" means is probably different from what Skyline thinks.
Skyline treats "B" as an amino acid whose mass is approximately 114 Daltons, which is almost certainly different from what your peptide search engine was assuming that "B" was.

I think things would work better if you told your peptide search engine that one of the standard amino acids had a variable modification on it whose chemical formula was what it needed to be to change that amino acid into the nonstandard amino acid that you are looking for.

Yes, Skyline does not allow you to specify that a modification applies to "B", "J", "X", or "Z", so it would probably not be possible to get any of those letters to work with your peptide search results.

Let me know if it is not possible for you to apply a modification to one of the standard amino acids instead of using "B".
We might be able to fix Skyline so that it works better with nonstandard amino acids.
I know in the Proforma v2 specification ( https://github.com/HUPO-PSI/ProForma/blob/master/SpecDocument/Release_v2.0/ProForma_v2_Final.pdf ) it says that the amino acid "X" should represent an amino acid of zero mass so that you can easily add a modification to it to represent a nonstandard amino acid. Skyline does not support this, but we could probably implement support for this in a few weeks.
-- Nick
 
lulmer responded:  2022-10-25
Thanks that file does work for me!

I've found that we get slightly different database search results when we run our searches as a mass shift on an existing amino acid instead of the additional amino acid ''B.' 'B' in our case is the crosslinking reagent benzoylphenelaylanine. There are very different results for crosslinked peptides when running searches with a mass shift on existing amino acids instead of an additional amino acid, so improved skyline compatibility for nonstandard amino acids would definitely be helpful for my use case.

Thanks!
Lindsey
 
Nick Shulman responded:  2022-10-25
By the way, I am not sure if you are familiar with the crosslinking features in Skyline:
https://skyline.ms/wiki/home/software/Skyline/page.view?name=Crosslinking

Skyline also understands some crosslinking aware peptide search results.
There are some crosslinking peptide search engines listed on this page:
https://proxl-ms.org/

Currently, the only crosslink search results that you can create a BiblioSpec library from are "byonic" and "kojak" (and you have to convert those results to .proxl.xml first) but if you needed support for a different crosslink peptide search engine we could probably add support for it easily.
-- Nick