view spectrum

support
view spectrum chia-lin  2019-03-11
 

Hi,skyline members

I build a peptide library with Maxquant results. I added all peptides by view library spectrum. But the spectrum did not show while the peptide list was ok. I will submit the image and hope to your reply. Thank you!

 
 
Nick Shulman responded:  2019-03-12
In the Spectral Library Explorer, you can see that some peptides do not have a symbol next to them.
The peptides without the symbol have modifications which do not match any of the modifications that you have told Skyline that you are using.

When you bring up the Spectral Library Explorer, Skyline tries to guess which modifications are being used by the library. Skyline usually shows you a list of the modifications that Skyline thinks are there and you can select all of the modifications and add them to your Skyline document.

The peptide sequence is A[?]AAVAAAGAGPLS[?]PEELVPK+++
Skyline needs to know what the modification is that is on that first A and the S.
The spectral library only stores the mass of the modification. If you hover your mouse over the peptide sequence in the Spectral Library Explorer I think Skyline will show you the mass of those modifications.

If, when you bring up the Spectral Library Explorer, Skyline does not suggest the correct modifications to you, you can instead tell Skyline about the modifications by going to:
Settings > Peptide Settings > Moidifications

It looks like you have already told Skyline that you have a heavy modification on all of your amino acids. This is why all of the peptides in your Targets tree are blue and bold face. Did you intend to do that?

If you would like, you can send us your Skyline document and we can figure out what is going wrong.

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

If that .zip file is less than 50MB, you can attach it to this support request.
Otherwise you can upload it here:
https://skyline.ms/files.url

If you want to learn more about modifications in Skyline, this webinar might be helpful:
http://skyline.ms/webinar10.url
-- Nick
 
chia-lin responded:  2019-03-13
Thanks for your kind help! I have submitted my document and the name is chia lin.
 
Nick Shulman responded:  2019-03-14
If you go to:
Settings > Peptide Settings > Modifications
you have a lot of modifications listed there which I think are incorrect, and you are missing a few modifications that should be there.
I am attaching a screenshot of what I think you should make your set of modifications be.
Note that only the Carbamidomethyl (C) modification has a checkmark next to it.
The other modifications are:
Phospho (ST)
Phospho (Y)
Acectyl (N-term)
Oxidation (M)

These unchecked modifications are "explicit modifications" which Skyline will only apply to a peptide if you explicitly tell Skyline to, or if Skyline sees the modification in a spectral library.

I hope this helps.
I believe Webinar 10 is our best resource for learning about modifications in Skyline:
http://skyline.ms/webinar10.url
-- Nick
 
chia-lin responded:  2019-03-15
thank you,Nick! The problem has been solved successfully.