Importing Transition List Containing Lysine Succinylation

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Importing Transition List Containing Lysine Succinylation aaron robinson  2019-03-12
 

Hi,

I'm not able to import a transition list into Skyline containing Lysine Succinylated peptides. See attached. I have the mod in my modifications and am importing it in as UniMod:64.

I'd like to publish a DIA dataset containing succinylated peptides and although it works fine if I use .pep.xml and use Skyline to build my DIA library I can't get it to go through importing a transition list to build my library.

Also attached is my sample transition list (1 peptide).

Please advise.

-Aaron Robinson

 
 
Nick Shulman responded:  2019-03-12
If you go to:
Edit > Insert > Peptides
and insert the peptide:
K(UniMod:64)WLPELVDR
you can get that peptide into your document.

If you right-click on the peptide in the Targets tree and choose "Pick Children", you can add the charge 3 precursor. (If the charge 3 precursor is not there, then push the funnel button to turn off the filter in the child picker.

Skyline thinks that the charge 3 form of that peptide has m/z 419.2276.
Your transition list has the precursor listed as 399.8923959.
Do you think a succinyl modification is different than what Skyline thinks it is?

-- Nick
 
aaron robinson responded:  2019-03-12
Hi Nick,

I think Skyline thinks it is a different mod with a different weight.

From Unimod.org

View     64    Succinyl    Suc_anh_light    Succinic anhydride labeling reagent light form (N-term & K)    100.016044    100.0728    H(4) C(4) O(3)

I think the mass might be off in Skyline.

I'm able to import a pep.xml file fine and it finds the succinylation because I've made it a mod in peptide settings but when I import a transition list I don't get that option to add mods that aren't in my document/etc.

Does that make sence?

-Aaron
 
Nick Shulman responded:  2019-03-12
Yes, that's the same formula that Skyline thinks that modification is.
I wonder where you are getting that 399.89.

Maybe you could zip up some of your .pep.xml and .mzML (or .mzXML) files and send them to us.

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

-- Nick
 
aaron robinson responded:  2019-03-13
Yeah...I get what you are saying. There must be some error in my library build step (Spectrast). I'll investigate on my end. The pep.xmL files import fine/there isn't an issue.

I appreciate the help!

-Aaron
 
aaron robinson responded:  2019-03-13
Hi Nick,

We were able to figure it out on our end. There was an issue with our spectrast2tsv.py settings.

Thanks for pointing us in the right direction!

-Aaron