How to set the diagnostic peaks of composition？

[Reedy91]

Thank you for developing the excellent tool fragpipe！
I am searching for phosphorylation at the H and Y sites, and I hope to improve the accuracy of site identification by tracking certain diagnostic ions. Could you please advise me on how to set the diagnostic peaks of composition (C5 H8 N3 O3 P) for H and composition (C8 H10 O4 N P) for Y?

[dpolasky]

Hi,
You can use our "detailed mass offset" search mode to search phosphorylation of H and Y only for spectra with those diagnostic ions above the minimum specified relative intensity (e.g., 0.05 = 5% of the base peak in the spectrum). There is a tutorial page here https://fragpipe.nesvilab.org/docs/tutorial_offset.html with information on how to set up the detailed offset search, and it includes an example template file with phosphorylation of Y defined with a diagnostic ion. You can add H as a separate row in the template to consider only the H-specific ion for H and the Y-specific ion for Y. You will need to compute the m/z of the formulas with the charging proton for diagnostic ions (so [C8 H10 O4 N P] + H+ = 216.042, rather than 215.035).

Depending on the data and how it was enriched, it is usually a good idea to also search phosphorylation as a variable modification on ST (in addition to the mass offsets) to avoid forcing the search engine to place the phospho on H/Y.

Best,
Dan