You are on the right track using "dehydro cysteine", assuming the peptide with the disulfide is a single polypeptide chain (and not a disulfide crosslink of two peptides, each with a single cysteine). I checked a bogus peptide FACEFACEK and made the two Cys's dehydro Cys and got the correct mass. Maybe you just need to re-import the data?

I too would greatly appreciate the ability to add disulfide linked peptides in Skyline, including intra-linked disulfides wintin one peptide as well as inter-linked disulfides between two or more peptides. This would be a helpful tool in my line of work (monitoring disulfide scrambling in pharmaceutical antibodies). Any update on if and when this will be an option in Skyline? THanks in advance.

Kind regards
Dan

Hi, Dan,

I have been working on an external tool for Skyline that will allow you to specify a peptide sequence as the modification on a particular amino acid. The tool calculates the chemical formula of that peptide modification, and then adds a peptide with that modification to your Skyline document.

Currently, this tool requires a special build of Skyline, and then, using that special build of Skyline, you have to install the external tool.
The special build of Skyline is at:
https://proteome.gs.washington.edu/~nicksh/rj8/newtoolservice/

And, when you are running that special build of Skyline, you can use:
Tools > External Tools > Add > From File
to add the external tool from this URL:
http://proteome.gs.washington.edu/~nicksh/rj8/CrossLinkerTool.zip

The build of Skyline that is up there is from a month ago. I'll try to put a newer version of that up there next week.
I will send you an email with a few more instructions about how to use the tool.

Hi Nick, thank you very much, much appreciated. I will test this tool when time allows early next year. Quick question: does this tool allows more than one disulfide linkage between two or more peptides? Thanks again. BR Dan

Dan,
No, the tool currently only allows one modification per peptide. I'll look into fixing this.

When you use the tool to insert a crosslinked peptide, the tool adds a modification definition to your document. After that modification has been added to the document, you can explicitly add that modification to other peptides.
So, if you want to put two modifications on a single peptide, you can use the tool to add the peptide with modification A, and add the peptide with modification B, and then, in Skyline, use the menu item:
Edit > Modify Peptide
to add the second modification to the peptide.

Resurrecting this old thread. Has this capability been added to either the current release of Skyline, or Skyline daily, or should we still use the special build? Thanks

Still have to use the special build.

While we are waiting: is it possible in Skyline to manually define precursor and fragment ion masses (e.g. for PRM), i.e. not to use precursor and fragments ion masses predicted from the defined amino acid sequence? This would really helpful, e.g. when working with complex disulfide complexes, such as IgG2, which results complexes with up 6 peptides linked by 6 disulphide linkages (IgG2 isoform B). Wishful thinking maybe...

There has been some progress on Skyline for metabolomics, where you can use edit/insert/transition list to pull up a window for inserting transition lists for non-peptide things. Click the small molecules radio button, and look through "columns" and "help". The idea is to be able to specify elemental compositions and/or m/z values of precursor and product ions (independent of peptide sequence, since this is meant for small molecules). If you went to the trouble of calculating the m/z or elemental comp for disulfide-linked peptides and fragments and inserting those numbers, then skyline can plot them out.

That is great information, rj8@uw.edu, thank you! I just tested the small molecules approach for disulphide complexes today, and it worked beautifully. All you need are the precursor and fragment masses (or elemental compositions), and this information can easily be obtained from e.g. GPMAW. Really cool, only wish I knew about this approach a year ago :)

Hello,

I would like to monitor by SRM a peptide with a disulphide bridge between two peptides. I haven´t been able set up the Skyline document for that. I have followed the instruction of downloading the special build and running the external tool (Crosslinker tool; see attached document) but I haven´t been able to insert the sequence of the 2 peptides bound by a disulphide bridge.

Can you help me with that please.

Thank you very much.

Best, Daniela.

The crosslinker tool does its work by defining in Skyline a pair of modifications that have the chemical formula of the two crosslinked peptides plus the crosslinker.
If you go to:
Settings > Peptide Settings > Modifications
you can see what the modifications that it has defined for you.

The crosslinker tool also adds two peptides to your document, one for the left and right peptides, that have these new modification on the specified amino acid position.

I cannot tell from your screenshot whether any of this is going wrong.
Is the problem that the modified peptide gets inserted into your document, but it is missing precursors or transitions? That might have something to do with your settings (Settings > Transition > "Filter" or maybe "Instrument"). You can always add precursors to a peptide in Skyline by hovering the mouse over the right edge of a peptide until an inverted triangle appears, and then clicking that triangle to choose children for the peptide.

It might be helpful if you could post your Skyline document.

In Skyline, you can use the menu item:
File > Share > (Complete) to create a .zip file containing your Skyline document and supporting files.

You can attach that .zip file to this support request, and I'll take a look at it.

Dear Nick,

Thank you very much for your help. I have attached the Skyline document. I would like to add two identical peptides crosslinked by a disulphide bridge (corresponding to the dimer form of the protein), to monitor by SRM the amount of dimer formation.

For that I will use Xevo-TQXS from Waters. I appreciate any help you can give me in setting up this document.

Best Regards,

Daniela.

I think you are almost there. I can see two things going wrong:

1. I believe that (not 100% sure) for a disulphide bridge, you want the crosslinker formula to be "-H2". In your PowerPoint screenshot, it looks like you had "-H", but you need to include the hydrogen lost from both peptides, so it's "-H2".

2. Your transition filter settings are preventing Skyline from giving you any precursors. You can fix this by going to:
Settings > Transition Settings > Filter
and adding "2,3" to the list of Precursor charges.

Another way to add precursors to your document and is to hover the mouse over the right edge of a peptide in the Targets tree, and click the inverted triangle which appears. This brings up the Child Picker for the peptide. You will need to click on the filter button on Child Picker, because by default, Skyline hides the precursors that do not match your filter settings.

Dear Nick,

Thank you very much for your help. I have followed your suggestion and I have changed the settings. Now I can see both precursor and transitions, but unfortunantely the m/z of the precursor does not match with the experimental one given by crosslink disulphide search engines. I have attached the new Skyline document. Do you think it is the case of just adding -H2 to the formula? How can I do that?

Best regards, daniela.

Yes, you just need to add -H2 to the formula of the modification.

That is, you go into:
Settings > Peptide Settings > Modifications
then
Edit List...
select the modification you want to modify
then
Edit
and change the Chemical Formula

The modification that you had been using was called "VACMPTFQFFKKGQKVGE-CC". In my screenshot here, I changed the name of the modification to "VACMPTFQFFKKGQKVGE-H2", and I also changed the chemical formula so that it was "C94H145N23O24S2-H2".

Then, after I hit OK on the peptide settings dialog, I needed to go and right-click on the Peptide in the targets tree, and choose "Modify", and apply this modification to the "C" amino acid in that peptide.

Dear Nick,

Thank you very much for your help. I have followed your suggestions and completed the Skyline document (attached). Unfortunantely the m/z of the precursor that contains a disulphide bond does not match with the search results obtained with the software (ppt attached). Could you help me with that?

Thank you very much for your help.

Best Regards, Daniela.

Sorry I did not attach the ppt in the previous message. The ppt is attached now.

Thank you very much.

Best Regards, daniela.

The crosslinker tool did not know that you wanted the crosslinked peptide to have an oxidized methionine.

You just need to modify the chemical formula in the definition for the modification "VACMPTFQFFKKGQKVGE-H2" and add an oxygen to it (in the .sky.zip that you sent me, it had 24 oxygens. If you change that to 25, then the M/Zs will match up more closely with what's in your PowerPoint).

Thank you very much for your help.
Best.

I am having a hard time of getting the correct sequence in for insulin. Tried to download the external tool and add the mods but keep getting an error!

In that error message that you are seeing, there is a lot more text in that textbox with the scrollbar in that dialog.

Can you select all of that text, copy it, and paste it into this support request? I will probably be able to figure out what is going wrong if I can see the rest of that message.

Kerry,

I think this might have something to do with your Skyline settings.
I would like you to send me your Skyline settings from the special "Skyline-daily-newtoolservice" that you have.
In that Skyline, you first use the menu item:
Settings > Save
and give the current settings a name.
After you have done that, you can use the menu item:
Settings > Share
to export the settings to a Skyline settings file ("*.skys").

Also, this bug might have something to do with the Skyline document that you are trying to insert the crosslinked peptides into, although I suspect that document might be blank.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .sky.zip file containing your Skyline document and whatever supporting files it might have.

Can you attach both the .skys and .sky.zip files to this support request?
(Alternatively, you can upload those files here: https://skyline.ms/files.url )

The external tool is having trouble asking Skyline for the list of modifications. I cannot think of any reason that would happen, but I might be able to figure it out after I see those files.

Here is my skyline file and settings that you requested!

Kerry,

The crosslinker tool only works with a special build of Skyline.
You have to install it here:
https://proteome.gs.washington.edu/~nicksh/rj8/newtoolservice/