Mind Your Spectra: Points to be Aware of when Validating the Identification of Isobaric Histone Peptidoforms
Mass spectrometry (MS) has become a central technique to identify and quantify post-translational modifications (PTMs), overcoming limitations of antibody-based methods. Histones get dynamically modified by diverse chemical groups, particularly on their numerous lysine residues, to fine-tune all DNA-templated processes. Reliable identification of histone PTMs remains challenging and still requires manual data 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 peptides with the same atomic composition. 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 software tools during the conversion of RAW files into peak lists, and highlighted how some parameters may erase the informative low-mass fragments. We established the fragmentation profiles and retention times for forty H3 K27-R40 variantxPTM combinations, including the mouse-specific variants H3mm7 and H3mm13, and targeted their detection in histone samples extracted from mouse testis and brain via a scheduled parallel reaction monitoring (PRM) analysis. The transcripts of these two mousespecific variants were reported to be highly abundant in these tissues and the corresponding proteins may seem to be identified by data-dependent analyses. However, we only detected very low levels of the unmodified form of H3mm7 and found no trace of H3mm13 by PRM. Our work contributes to reliably deciphering the histone code shaped by distinct sequence variants and numerous combinations of PTMs.