|Structural and isotope-heavy modifications occurring on the same residue||lpino||2021-06-30|
Hi team! I've been banging my head against this one for a little while, so I thought I'd reach out to see if you can save me from any more headache!
Context: histone PTM analysis with D3-acetyl chemical derivatization. This means peptides will have biological (structural) modifications (including "normal" acetyl), and then any unmodified lysines and peptide N-termini should be D3-acetylated.
I did a paired DDA+DIA for each sample. Searched the DDA with Byonic (PD), built a spectral library with Bibliospec (Skyline), and added the detected peptides to the Skyline doc for quant by DIA XIC (Library Explorer > Associate proteins > Add).
Okay here's where I'm getting turned around. I am getting the craziest permutations of modification + D3 in the target list, and can't for the life of me interpret them! Some of these peptides have the same residue doubly-modified, e.g. a lysine with both structural "Acetyl" and then also isotope heavy "Acetyl:2H(3)". They all have a library spectra associated with them, so is this perhaps Skyline forcing all lysines to be "Acetyl:2H(3)" in addition to any other structural modification they might have?
I have no idea what I did wrong, but this is admittedly the deepest foray I've ever made into PTM analysis and I'm probably hacking some features together to get this to work. Is there some other way I should set up my Peptide Settings > Modifications? Any ideas or help would be greatly appreciated! Minimal Skyline doc example attached.