Lysine and Arginine Protein Post-translational Modifications by Enhanced DIA Libraries: Quantification in Murine Liver Disease

Proteoforms
containing post-translational modifications (PTMs)
represent a degree of functional diversity only harnessed through
analytically precise simultaneous quantification of multiple PTMs.
Here we present a method to accurately differentiate an unmodified
peptide from its PTM-containing counterpart through data-independent
acquisition-mass spectrometry, leveraging small precursor mass windows
to physically separate modified peptidoforms from each other during
MS2 acquisition. We utilize a lysine and arginine PTM-enriched peptide
assay library and site localization algorithm to simultaneously localize
and quantify seven PTMs including mono-, di-, and trimethylation,
acetylation, and succinylation in addition to total protein quantification
in a single MS run without the need to enrich experimental samples.
To evaluate biological relevance, this method was applied to liver
lysate from differentially methylated nonalcoholic steatohepatitis
(NASH) mouse models. We report that altered methylation and acetylation
together with total protein changes drive the novel hypothesis of
a regulatory function of PTMs in protein synthesis and mRNA stability
in NASH.