Mind Your Spectra: Points to be Aware of When Validating the Identification of Isobaric Histone Peptidoforms.

Abstract

Mass spectrometry has become central to identifying and quantifying histone post-translational modifications (PTMs), surpassing limitations of antibody-based methods. Histones are dynamically modified by multiple structures, especially at lysine residues on their N-terminal tails, to regulate DNA-templated processes. Reliable identification of histone PTMs remains challenging and still requires manual curation. This study focused on the Lys27-Arg40 stretch of histone H3, considered four sequence variants, an increasing number of lysine PTMs and artifacts coming from histone sample processing, which resulted in many isobaric peptides. Our analysis revealed the value of low-mass b1 and cyclic immonium fragment ions to validate identification of the distinct peptidoforms. We examined how MS/MS spectra are transformed by common identification software during the conversion of raw files into peak lists, and highlighted how some parameters may erase the informative low-mass fragments. We targeted the detection of 40 H3 K27-R40 variant × PTM combinations, including the mouse-specific variants H3mm7 and H3mm13, in histone samples extracted from mouse testis and brain via a parallel reaction monitoring analysis. We only detected very low levels of unmodified H3mm7. Our work contributes to reliably deciphering the histone code shaped by distinct sequence variants and numerous combinations of PTMs.