Optimization of the analysis of post-translational modifications on histones using LC-MS with ion mobility
Hogeschool West-Vlaanderen
2024
Core histones (H2A, H2B, H3, and H4) form nucleosomes, the basic units of chromatin, which compact DNA into chromatin fibers. Euchromatin, with loosely packed structure, facilitates transcription by allowing enzyme access to DNA, whereas heterochromatin's tight packing blocks transcription. Histone proteins contain tails rich in lysine and arginine residues, which undergo various posttranslational modifications (PTMs). These modifications influence the regulation of chromatin structure and gene activity. Post-translational modifications, such as histone lysine methylation, acetylation and phosphorylation are prevalent in various cancer types. The aim of the study is to detect and localize specific PTMs on histones by optimizing the existing method for nuclei isolation and sample purification. The research employs the OPM-2 cell line, for which sample preparation begins with the isolation and breakdown of the nuclei. Next, magnetic beads are utilized to purify the sample by binding the proteins and removing contaminants, followed by overnight digestion. After preparing the Evotips, the samples are injected into the Evosep One connected to the timsTOF HT for tandem MS analysis with trapped ion mobility. The addition of an extra washing step during nuclei isolation, which was tested during protocol adjustments, had no effect on the purity of the sample. Similarly, the separate addition of SDS and Benzonase during nuclei isolation showed no positive impact. When the effect of an additional protein purification step using 5 µl RPW cartridges was investigated, it led to a significant improvement in purity, with a lower total protein yield and histones being the most abundant proteins in the sample. With the latest optimization, nine peptides of interest on H3 could be detected. However, the evaluation of this optimized protocol is still ongoing and further optimization remains necessary.
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