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Filter for N-glycans

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Filter for N-glycans sarah shapley  2025-11-11 13:18
 

Hi there!

I'm hoping you can help me with a problem that's hopefully somewhat straightforward. I'm trying to filter the skyline document for N-modified residues resulting from a FragPipe Glyco-N-DIA search (with a DDA library). To do so, I'm going to Peptide Settings > Filter > Exclude peptides containing: NXT option. However, I'm noticing this doesn't function as expected since my peptide/precursor/transition numbers stay the same.

Overall, I'm hoping to find an ability in Skyline to filter my target list to those with an N-modification (doesn't have to match the motif). Please let me know if there are any questions!

Thanks again for all the work you do for the greater community!
 
 
Juan C. Rojas E. responded:  2025-11-12 01:57

Hi Sarah,

Here is an alternative solution:

  • Create Document Grid report that has the Peptide Modified Sequence column
  • Apply a filter in the column for the string "N[". To remove all peptides that are not N-glycosylated then use the "Does not contain" filter condition
  • Once filter is applied select all rows and delete (using X symbol)
  • Remove filters

At the end of this you would have all peptides that have modifications on N residues. However, this might also contain other N modifications if you searched for it. This approach does not consider the sequence motif and relies on the search engine (e.g. MSFragger Glyco) using the motif for peptide glycopeptide proposal.

It would be neat if Skyline can use this N-glycan sequence motif, but it would have to be extended to both Threonine and Serine, i.e. N-X-S/T.

I hope it helps!

Sincerely,
Juan C.