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a-ions from fully SIL labelled amino acids

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a-ions from fully SIL labelled amino acids jfoe  2024-02-14 04:35
 

Dear Skyline-Team,

I have a workflow where I create spectral libraries from Skyline. I recently noticed that I am missing some a-ions from my SIL-labelled peptides, if the fragmentation is next to the SIL label.

The reason is, that while the common b- and y- ions fragment neatly at the peptide bonds, the a-ion loses a C-atom that belongs to one of the adjacent amino acids.
Skyline always subtracts a 12C. When the amino acid is fully SIL, this should be a 13C. I suspect this is tough to implement in a general sense but it is very common that SIL are fully labelled so maybe skyline could implement an exception where a 13C is removed if there is no 12C at all in the amino acid.

I am including a screenshot that hopefully shows you why this causes me to lose signal. In that specific case there is actually some peak at the wrong a6+, too, but I am missing the main, correct a6+ peak. In effect, this is quite bad because the spectral library will end up with a bad dotp when detecting the natural non-labelled peptide because it expects only a very weak a6+ ion.
Also, I am including the skyline file.
Skyline version is 23.1.0.380 x64 on Windows.

Let me know if anything is unclear.

Best wishes,
Jonas
 
 
Nick Shulman responded:  2024-02-14 05:07
You might be able to achieve what you need if you tell Skyline that your modification has a potential neutral loss whose chemical formula has a heavy carbon.
An "a" ion fragment usually has a chemical formula which is the b ion with a loss of "CO".

You could tell Skyline that your modification has a neutral loss whose formula is "C'O" (C' is the symbol that Skyline uses for heavy carbon), and then add to your document b ions which also have a 29 Dalton loss.
(alternatively, you could say that the chemical formula for the loss was "C'-C" (i.e. loses a heavy carbon and gains a light carbon) and then use the "a" ions with a neutral loss of "1".)

By the way, it sounds like you intended to attach a Skyline document, but no document was attached. Sometimes this happens if the file you were trying to attach is greater than 50MB which is the maximum size of an attachment on the support board. If you would like to send us larger files you can upload them here:
https://skyline.ms/files.url
-- Nick
 
jfoe responded:  2024-02-14 05:25
Dear Nick,

thanks for the swift response.
Trying this out, I notice that I cannot set neutral losses for isotope modifications (see: skyline_a6_wrong_I_label_mod.png).

I usually create the spectral library in skyline using these isotope labelled peptides and then skyline knows to translate the masses when I am targeting light peptides.
I am concerned that if I was using a workaround like this, the matching wouldn't work. I suspect skyline would definitely need to identify this as a6.

I am uploading also the skyline file that I accidentally missed the upload of.

Cheers,
Jonas
 
Nick Shulman responded:  2024-02-14 06:06
The "Ion Match Tolerance" at "Settings > Transition Settings > Library" controls how close the observed m/z in the library spectrum needs to be to the calculated m/z of the fragment ion for Skyline to consider them to be a match.
You currently have that set to "0.001".
You might be able to improve things if you set that to a larger number such as "1" so that the 1 Dalton difference caused by the heavy carbon atom would not prevent Skyline from thinking recognizing it as matching the a6 transition.
-- Nick
 
jfoe responded:  2024-02-15 08:22
Dear Nick,

thank you for the input.
I was able to specify a special ion for that peptide, which I called "RAHYNiVTF - a6+". That one matched exactly the peak of interest.
When importing the resultant spectral library, I was not able to make it match the light a6+ to the special ion. I think this is because of the max. tolerance of 1 m/z (see a6_special_ion_speclib.png). In that screenshot you can also see the spectral library displayed, which shows the true special ion a6+ vs the
mismatched a6+.
I think increasing the m/z limit would create many issues for me because ions would start getting mismatched (e.g. water loss -18: vs ammonia loss: -17).

I am also including the skyline zip for reference.

Best wishes,
Jonas