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How to add acetylation modification to the K-terminal in Skyline document?

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How to add acetylation modification to the K-terminal in Skyline document? caixue  2024-06-13 01:11
 

I have identified several K-acetylated peptide sequences in the Fragpipe analysis of my data. I would like to see the peak groups and spectra of these peptide sequences in Skyline.

The peptide segment acetylated on the K-terminal is shown below：
ALAAGGYDVEK[42.0106]NNSR
GK[42.0106]YYAVNYPLR
IEGEGSVLQAK[42.0106]LK
ISIC[57.0215]SSDK[42.0106]R
NGLSLAALKK[42.0106]
NGLSLAALK[42.0106]K
n[42.0106]SETAPAETATPAPVEK[42.0106]SPAK

I insert the peptides as below:
ALAAGGYDVEKNNSR
GKYYAVNYPLR
IEGEGSVLQAKLK
ISICSSDKR
NGLSLAALKK
SETAPAETATPAPVEKSPAK

And I also set modifications in peptide setting as attached figure 1. However, the peptide in the document failed to display the acetylation marks, as illustrated in attached figure 2.
I don't know where the operation went wrong.
I look forward to your reply.
Best wishes.
 
 
Nick Shulman responded:  2024-06-13 05:02
In your "Edit Structural Modification" screenshot, "K" is selected in the "Amino Acid" dropdown and "N" is selected in the "Terminus" dropdown.
A modification defined in this way would only be allowed to be applied to K amino acids which are also at the N terminus of the peptide, which does not seem to be what you need.
In Skyline you would need two separate modifications: one modification which can be applied to any "K" amino acid regardless of where it appears in the peptide sequence, and modification which can be applied to an N-terminus amino regardless of which amino acid letter that is.

Skyline calls these two modifications "Acetyl (K)" and "Acetyl (N-term)" and if you select those names in the "Edit Structural Modification" dialog, Skyline will fill in the rest of the settings with the appropriate values.

If you would like to learn more about working with modifications in Skyline the following webinar is always recommended:
http://skyline.ms/webinar10.url
-- Nick