Mass spectrometry peptide analyses of Figure 2 gel slices
Description
A giant virus genome is densely packaged by stable nucleosomes within virions. The two doublet histones of Marseillevirus are distantly related to the four eukaryotic core histones and wrap 121 basepairs of DNA to form remarkably similar nucleosomes. By permeabilizing Marseillevirus virions and performing genome-wide nuclease digestion, chemical cleavage and mass spectrometry assays, we find that the higher-order organization of Marseillevirus chromatin fundamentally differs from that of eukaryotes. Marseillevirus nucleosomes fully protect DNA within virions as closely abutted 121-bp DNA wrapped cores without linker DNA or phasing along genes. Likewise, we observed that nucleosomes reconstituted onto multi-copy tandem repeats of a nucleosome positioning sequence are tightly packed. Dense promiscuous packing of fully wrapped nucleosomes rather than “beads-on-a-string” with genic punctuation represents a new mode of DNA packaging by histones. We suggest that doublet histones have evolved for viral genome protection and may resemble an early stage of histone differentiation leading to the eukaryotic octameric nucleosome. After silver-staining SDS-PAGE, protein from gel slices was extracted for MS analyses, providing trypsinized peptides matched to Marseillevirus predicted protein sequences and ordered based on number of peptides recovered.
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Steps to reproduce
1) Resolve proteins by 4-20% SDS-PAGE. 2) silver-stain using the Pierce kit (Thermo cat. no. 24600). 3) Excise protein bands, destain, and proteolytically digest. 4) Desalt proteolytic peptides and analyze by LC-ESI-MS/MS using a ThermoScientific Orbitrap Fusion mass spectrometer. 5) Analyze ,ass spectra with ThermoScientific Proteome Discoverer version 2.5 software using the Marseillevirus marseillevirus translated protein sequences from NCBI (ref. sequence NC_013756.1) appended to the cRAP (https://www.thegpm.org/crap/) common contaminant protein database. 6) Filter peptide identification results to a false discovery rate