A Super-SILAC Approach for Profiling Histone Posttranslational Modifications

Noberini, Roberta; Longhi, Elisa; Bonaldi, Tiziana

doi:10.1007/978-1-0716-2863-8_7

Cited by 3 publications

(1 citation statement)

References 24 publications

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“…Histones were extracted from 5 to 10 tissue sections of 10 µm thick or 2 million cells as previously described. 42 Approximately 4 µg of histone octamer were mixed with an equal amount of heavy-isotope labeled histones, which were used as an internal standard, 43 and separated on a 17% SDS-PAGE gel. Histone bands were excised, chemically acylated with propionic anhydride and in-gel digested with trypsin, followed by peptide N-terminal derivatization with phenyl isocyanate (PIC).…”

Section: Methodsmentioning

confidence: 99%

Mass Spectrometry-Based Profiling of Histone Post-Translational Modifications in Uveal Melanoma Tissues, Human Melanocytes, and Uveal Melanoma Cell Lines – A Pilot Study

Herwig-Carl,

Sharma,

Tischler

et al. 2024

Invest. Ophthalmol. Vis. Sci.

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Purpose Epigenetic alterations in uveal melanoma (UM) are still neither well characterized, nor understood. In this pilot study, we sought to provide a deeper insight into the possible role of epigenetic alterations in the pathogenesis of UM and their potential prognostic relevance. To this aim, we comprehensively profiled histone post-translational modifications (PTMs), which represent epigenetic features regulating chromatin accessibility and gene transcription, in UM formalin-fixed paraffin-embedded (FFPE) tissues, control tissues, UM cell lines, and healthy melanocytes. Methods FFPE tissues of UM ( n = 24), normal choroid ( n = 4), human UM cell lines ( n = 7), skin melanocytes ( n = 6), and uveal melanocytes ( n = 2) were analyzed through a quantitative liquid chromatography-mass spectrometry (LC-MS) approach. Results Hierarchical clustering showed a clear separation with several histone PTMs that changed significantly in a tumor compared to normal samples, in both tissues and cell lines. In addition, several acetylations and H4K20me1 showed lower levels in BAP1 mutant tumors. Some of these changes were also observed when we compared GNA11 mutant tumors with GNAQ tumors. The epigenetic profiling of cell lines revealed that the UM cell lines MP65 and UPMM1 have a histone PTM pattern closer to the primary tissues than the other cell lines analyzed. Conclusions Our results suggest the existence of different histone PTM patterns that may be important for diagnosis and prognosis in UM. However, further analyses are needed to confirm these findings in a larger cohort. The epigenetic characterization of a panel of UM cell lines suggested which cellular models are more suitable for epigenetic investigations.

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Section: Methodsmentioning

confidence: 99%