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disulfide crosslinked homodimeric also covalently modified peptide does not appear to work

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disulfide crosslinked homodimeric also covalently modified peptide does not appear to work warham  2024-05-14 08:08
 

So this will probably go to Nick,

So disulfide homodimeric peptides load beautifully through the modify peptide interface. And crosslinks between different peptides can also take other chemical modifications in the insert peptide interface. On a mAb there is this sequence:

THTCPPCPAPELLGGPSVFLFPPKPK

And in evaluating it's disulfide integrity, this peptide

THTCPPC[+57]PAPELLGGPSVFLFPPKPK-THTCPPC[+57]PAPELLGGPSVFLFPPKPK-[DSS@4,4]

would be useful but I get the error that the peptide is not recognized by the software. I tried adding another copy of the sequence but there is no field for specifying different proteins in the same peptide entry on the insert peptide field. Also the insert peptide field does not seem to support multiple crosslinks between two peptides which does happen with disulfides.

Is there a way to do this that I'm missing? If it's too specific an issue, that's fine. But I thought that since there are a fair number of disulfide oligomers, it might affect others too.

Best,
Lance
 
 
Nick Shulman responded:  2024-05-14 09:17
I am not sure that I understand your question.
Where are you seeing an error about the peptide not being recognized?

I am able to insert a crosslinked peptide like that using the "Edit > Insert > Peptides" menu item.

Can you send me your Skyline document? In Skyline you can use the menu item:
File > Share
to create a .zip file containing your Skyline document and supporting files including spectral libraries.
Files which are less than 50MB can be attached to these support requests. You can always upload larger files here:
https://skyline.ms/files.url

It might also help if you could post a screenshot of what you are seeing.
-- Nick
 
warham responded:  2024-05-14 09:47
Hi Nick,

Thank you for looking at this. Here is the simplest possible case and the error I see (the .png)

Best,
Lance
 
Nick Shulman responded:  2024-05-14 10:13
This works for me.
In order to be able to insert a crosslinked peptide that uses the "DSS" crosslinker, that crosslinker would need to be listed in the "Structural modifications" list at "Settings > Peptide Settings > Modifications".
Skyline does not inherently know about any crosslinked modifications so in order to be able to use one in a Skyline document, the modification would already need to be listed in the settings, and the "Crosslinker" checkbox needs to be checked.

I cannot tell whether you have that crosslinker correctly defined because modifications which do not have a checkmark next to them only get included in the .sky file if at least one peptide in the document is using it.
I'm attaching a picture of what my DSS modification looks like.
-- Nick
 
warham responded:  2024-05-14 18:26
Hi Nick,

I got that to work with my xl named properly. However, as soon as I try to do a dimer peptide from a homodimeric protein where there is only one sequence, I still get denied.

Best,
Lance
 
Nick Shulman responded:  2024-05-14 18:45
Now it is telling you that the peptide sequence cannot be found in the protein that you are saying it is part of.

The leftmost peptide in the crosslinked peptide needs to be found in the protein sequence.

You can prevent this error by leaving the "Protein" column blank.
--Nick