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

ABSTRACT: Mass spectrometry (MS) is now well recognized as a powerful technique to identify and quantify post-translational modifications (PTMs), overcoming many of the limitations of antibody-based methods. Histones, which play a central role in all DNA-templated processes, are regulated by a wealth of dynamic modifications, particularly on their numerous lysine residues. Reliable identification of histone PTMs remains challenging and still requires manual data curation. In this study, we focused on the Lys27-Arg40 stretch of histone H3 and considered four sequence variants, an increasing number of lysine PTMs, and chemical artefacts coming from the specific protocol of 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 for validation of the identification of the distinct peptide forms. We examined how MS/MS spectra are transformed by several 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 variant×PTM 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. These two mouse-specific variants were reported to be highly abundant at the transcript level in these tissues and may seem to be identified at the protein level by data-dependent MS acquisition. 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.

INSTRUMENT(S):

ORGANISM(S): Mus Musculus (mouse)

TISSUE(S): Testis, Brain

SUBMITTER: Hassan Hijazi  

LAB HEAD: Delphine Pflieger

PROVIDER: PXD057347 | Pride | 2025-04-22

REPOSITORIES: Pride