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Precursor Ions Richard Lam  2019-03-28 17:10
 

Hello,

I was trying to use Skyline to predict the fragments ion of a known peptide.
I used the 'Import Peptide List' function and with the Transition Settings as shown in the attachment.
After the MRMs were generated in Skyline, I exported the MRM parameters as .csv by clicking 'File', 'Export', 'Report', then choose 'Peptide Transition List'.
Please see attachment for the report output.
The peptide is LCVLHEK, its neutral precursor is 822.4 and I expect the 2+ ion is at 412.2.
However, in the report, the peptide is shown as 'LC[+57]VLHEK' and the 2+ precursor ion is at 449.74.
Do you know why? Did I do something wrong in the settings?

Thanks!

Richard
 
 
Brian Pratt responded:  2019-03-28 17:32

Hi Richard,

What does your Peptide Settings > Modifications tab look like? You probably have some settings there that you don't actually want.

Thanks for using the Skyline support board!

Brian Pratt
 
Brendan MacLean responded:  2019-03-28 20:11

Yeah. That C[+57] is Carbamidomethyl (C). If you don't want that, you can uncheck this modification in the Peptide Settings - Modifications tab, but if I may ask how did you treat your Cysteines? We don't usually see completely unmodified Cysteine. Usually it is either Carbamidomethyl (C) [+57] or Carboxymethyl (C) [+58].

If you are sure you have bare unmodified Cysteine though, then it is easy enough to uncheck that modification.

--Brendan
 
Richard Lam responded:  2019-04-01 10:41

I just performed traditional protein digestion protocol, i.e. reduction by DTT, alkylation by IAA, then digestion by trypsin.
Do you think I should consider Carbamidomethyl (C) [+57] and/or Carboxymethyl (C) [+58] modifications? Thanks!

Regards,
Richard
 
Mike MacCoss responded:  2019-04-01 15:00

Hi Richard,
It depends. Are you alkylating with iodoacetamide or iodoacetic acid? Alkylating with iodoacetamide results in carbamidomethyl cysteine [+57] and alkylating with iodoacetic acid results in caboxymethyl [+58]. BTW, most people these days use iodoacetamide.

I hope this helps,
Mike
 
Richard Lam responded:  2019-04-02 12:48

Got it. I am using iodoacetamide. Thanks for the clarification.

Regards,
Richard