Heavy Modifications Label:13C(6)15N(2) (C-term K) and Label:13C(6)15N(4) (C-term R) from PLGS Final Fragment File Recognized as 13C

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Heavy Modifications Label:13C(6)15N(2) (C-term K) and Label:13C(6)15N(4) (C-term R) from PLGS Final Fragment File Recognized as 13C dwwilk02  2021-10-25
 

Hi,
I've been trying to build a library in Skyline 21.1.0.278 from a Waters PLGS 3.0.2 final fragment file (Waters MSe data from Synapt G2-Si), but have not been able to get Skyline to identify the heavy labels Label:13C(6)15N(2) (C-term K) and Label:13C(6)15N(4) (C-term R) correctly. These PTMs are identified as 13C in my attempts to build the library. The original string for the peptide.modification from PLGS in the attached file was Label%3A13C%286%2915N%282%29+%28K%29(15), but Skyline does not recognize this. I tried to modify it, but have not been able to get the correct PTM. Thanks for your help,
Daniel Wilkey

 
 
Brian Pratt responded:  2021-10-25

We'll have a look at this, thanks.

In the meantime, what's that non-ASCII character (hex code 0xB0) that shows up in the peptide.MatchedProductsString and fragment.str columns? Probably not related to the problem, but it's unusual.

Thanks for using the Skyline support board!

Brian Pratt

 
dwwilk02 responded:  2021-10-25

Hi Brian,
Thanks for your response and offer to help. I believe that character is an indicator of a fragment with a water neutral loss (e.g., b5° indicates a b5 ion with water neutral loss). Thanks,
Daniel Wilkey

 
Brian Pratt responded:  2021-10-25

It would be helpful to see the original file, but looking at the code, it seems the parser is expecting something more like "SILAC 13C(6) 15N(2)" than "Label:13C(6)15N(2)". I wonder if something has changed in PLGS and the way it represents these modifications?

 
dwwilk02 responded:  2021-10-25

Thanks Brian:) In the test file I sent earlier, Skyline was able to recognize the the modification when I changed it to "SILAC 13C(6) 15N(2)+K(15)". The original final fragment file is attached.
Daniel Wilkey

 
dwwilk02 responded:  2021-10-25

I possibly added the mods Label:13C(6)15N(2) (K) and Label:13C(6)15N(4) (R) into our version of PLGS. I used them instead of the SILAC mods in PLGS for the search. Thanks for your help with this,
Daniel Wilkey

 
Brian Pratt responded:  2021-10-25

Ah, OK. It's hard to know what to do with user-created values.

Thanks

Brian