Peptide Transitions

support
Peptide Transitions iej4  2019-06-19
 

I am setting up an MRM peptide quantitation method but I need to input a non-standard transition. I have a short proline containing peptide that is producing what seems to be a strong y-18 ion but Skyline doesn't display that fragment as a possibility so I can't use it in the method. Is there a way to change the fragmentation rules of skyline so that the fragment is allowed or to manually add a transition to a peptide?

Thanks.

 
 
Nick Shulman responded:  2019-06-19
You can right-click on the precursor in the Targets tree and use the menu item "Pick Children" to choose that transition.

You might need to press the filter button (it looks like a funnel) on the child picker popup window in order to be able to select that particular ion.

If this does not work, or if there's something more special about the y-18 ion that you are trying to add, then it might be helpful if you sent us your Skyline document so we can see what is going on.
In Skyline, you can use the menu item:
File > Share > (complete)
to create a .zip file containing your Skyline document and supporting files.

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

(But, probably the "Pick Children" menu item will work for you and you don't need to send us your file).
-- Nick
 
iej4 responded:  2019-06-19
Thanks for the response Nick. I have already tried the pick children option. The ion that I am seeing in the spectra that I believe to be a y-18 ion is not listed in the fragment ion list of the pick children dialog. Based upon standard fragmentation rules for y-18 ions, this peptide should not be producing a y-18 ion. That's why I was asking about changing the fragmentation rules are manually adding the transition. I tried to add the transition through the insert transition route but skyline rejects the transition because it does not recognize the fragment ion (in this case I believe it is a y5-18 ion).
 
Brendan MacLean responded:  2019-06-19
I assume by y5-18 you mean a water loss. To enable water loss ions, you need to go to the Peptide Settings - Modifications tab and add the modification "Water Loss (D, E, S, T)" modification which you can find at the bottom of the list of names of available modifications, once you click Add.

Make sure this modification is checked, and you should see the y5-18 transition in the list of available transitions.

There is also an "Ammonia Loss (K, N, Q, R)" in case you are interested in that as well.

Hope this helps. Good luck with your experiment.

--Brendan
 
iej4 responded:  2019-06-19
The peptide is LVPGFGK which does not contain any of the residues that typically result in a loss of water (DSTE). I am not sure why I am getting this loss of water fragment but I am. I have attached the ms/ms from a low res ion trap scan (Qtrap 5500). y5 is the strongest fragment signal followed by an ion 18 Da lower and then another ion that is also just shy of 18 Da lower (17.992 Da) - the last can be assigned as a5-NH3. Without a D,S,T or E in the sequence, Skyline won't let me select y-18 fragment. This spectra was generated using a pure synthetic peptide. To me it looks like the proline allows a neutral loss of water. Skyline seems to require DEST in the sequence for a neutral loss of water to occur.
 
iej4 responded:  2019-06-21
Brendan,

You gave me a lead to help solve this problem. I created a new modification under the Peptide Settings for "Water Loss (P)" for the proline residue. Now I can select the y5-18 ion no problem for my peptide. Thank you. That was a big help.

-Matt