Characterization of Histone post-translational modifications using a Top-Down, label-free, LC- MALDI-TOF Mass Spectrometry approach.

Introduction.The post-translational (PTMs) modifications of nuclear histones, as well known, play an essential role both in the DNA packaging mechanism in chromosomes that in the regulation of gene expression in different cellular processes, especially in response to molecular agents of environmental origin (epigenetic regulation). Aim.The characterization of histone PTMs is traditionally performed by the use of antibodies. However, these methods are often not sufficiently specific and today systems that use mass spectrometry are increasingly used. In this context, our research aims to improve the methodologies based on mass spectrometry mainly in relation to the comprehensivity, simplicity and rapidity of the characterization. Methods.For this purpose, a top-down analysis of MALDI- In Source Decay (MALDI-ISD) analysis, on HPLC fraction of nuclear extracts obtained from immortalized EBV lymphocytic lines, was applied. The cells grown in RPMI 1640 medium were lysed and the collected nuclear fraction was subjected to acid extraction with 0.8M HCl. A preliminary MALDI-TOF profiling analysis was performed on the whole crude extract, and successively this was subjected to HPLC separation from which homogenous fractions of histones (H2A, H2B, H3.1, H3.2, H3.3 and H4) were obtained. Fractions of interest were analyzed using MALDI-ISD using 1.4 DAN as matrix, in order to obtain sequencing and PTMs data. In some cases, a PSD analysis was also performed on fragments generated by the ISD (MS3) to confirm data. Results.The MALDI-TOF profiling of the crude nuclear extract revealed the presence of the principal types of histones, allowing also a relative quantitative evaluation. Subsequently, the MALDI-ISD analysis of the HPLC fractions provided information on both the amino acid sequence and the PTMs present. In particular, we were able to define, in the same experiment the presence in H4 of a dimethylation (above 90%) in the lysine at position 20 (H4K20Me2) Conclusions.In conclusion, the MALDI-ISD methodology allows to obtain global information on histone PTMs with specificity, rapidity and simplicity, effectively contributing to reveal the influence of environmental molecular factors