Development of a multiple reaction monitoring (MRM)-based LC-MS/MS method for the quantification of post-translational modifications on histone H3 variants in <i>Arabidopsis thaliana</i>

<i>Background</i>: although the canonical histone H3.1 and its variant H3.3 differ by only four amino acids, they exhibit distinct genome-wide binding patterns and regulate different biological pathways. Post-translational modifications (PTMs) on histone tails mediate diverse downstream regulatory processes, raising the question of whether H3.1 and H3.3 harbor variant-specific modifications. However, the minimal amino acid differences between H3.1 and H3.3 make it challenging to distinguish and quantify them using traditional methods. <i>Results</i>: in this study, we developed an integrated multiple reaction monitoring (MRM)-based LC-MS/MS method to accurately differentiate and quantify K27 and K36 modifications on H3.1 and H3.3 in <i>Arabidopsis thaliana</i>. Our findings show that H3.1 contains more K27 methylation marks, associated with gene silencing, whereas H3.3 is enriched in K36 methylation, a mark of active transcription. Additionally, we compared K36 methylation levels in wild-type and SDG8-depleted cells, revealing that the K36 methyltransferase SDG8 shows a strong preference for H3.3 in both <i>in vitro</i> and <i>in vivo</i> assays. By analyzing public datasets, we further identified a strong correlation between H3.3 and the regions where H3K36me3 levels were reduced in <i>sdg8</i> knockout cells. <i>Significance</i>: the MRM-based LC-MS/MS method established in this study provides a reliable and robust tool for the quantification of histone H3.1 and H3.3 PTMs in <i>Arabidopsis thaliana</i>. We demonstrate that the methyltransferase SDG8 shows a strong substrate preference for H3.3. This discovery highlights the importance of histone variant-specific modifications and suggests new avenues for research into their regulatory roles.