Computational protocol: Mitotic post-translational modifications of histones promote chromatin compaction in vitro

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Protocol publication

[…] Raw files were analysed with MaxQuant proteomics suite of algorithms (v. 1.5.3.17) [], integrated with the search engine Andromeda [].The data were searched against a chicken histone database (generated from a chicken proteome database downloaded on 09.10.2015 from UniProt) with the following parameters: the maximum allowed mass deviation was set to 4.5 ppm for precursor ions and 20 ppm for fragment ions; the minimum peptide length was set to 6 amino acids; the maximum number of missed cleavages was set to 5 with the maximum charge state 6; multiplicity was set to 2 with Lys8/Arg10 as the heavy label and max. labelled AAs were set to 7. Variable modifications included acetylation (Protein N-term and K), methylation (KR), di-methylation (KR), tri-methylation (K), phosphorylation (STY), GlyGly (K)—ubiquitylation remnant on lysine residue after trypsin digestion—and propionylation (K). FTMS top peaks per 100 Da were set to 20.In-house scripts were used to perform bioinformatic analysis of the MaxQuant processed data. Briefly, for c, the ‘ProteinGroups.txt’ table was loaded and filtered for ‘Potential contaminant’ ≠ ‘+’, ‘Reverse’ ≠ ‘+’, ‘Only identified by site’ ≠ ‘+’ and ‘Razor+unique peptides’ > 2. Intensities and ratios were log10 and log2 transformed, respectively. For c, ‘Acetyl (K)Sites.txt’, ‘Methyl (KR)Sites.txt’, ‘2Me (KR)Sites.txt’, ‘3Me (K)Sites.txt’, ‘Phospho (STY)Sites.txt’ and ‘GlyGly (K)Sites.txt’ tables were loaded and filtered for ‘Potential contaminant’ ≠ ‘+’, ‘Reverse’ ≠ ‘+’, ‘Localization probability’ > 0.95 and ‘Score’ > 80. Remaining sites were further validated in a manual fashion. Sites not supported by more than two confident spectra (Score > 80 and correct assignment of the modification) were discarded. An additional filtering step was performed. As depicted in c, H3T3ph, H3S10ph and H3S28ph marks are dramatically increased in mitotic cells. Therefore, interpretation of the SILAC ratio of any mark coexisting with the above-mentioned modifications is difficult to interpret. For instance, most of the evidences (and all of them in some cases) for H3R2(me), H3K4(me), H3K4(me2), H3R8(me2), H3K9(ac), H3K9(me), H3K9(me2), H3K9(me3), H3K14(ac), H3R26(me2), H3K27(me), H3K27(me2), H3K27(me3), H3S31(ph) and H3K36(me2) are from peptides that also contain H3T3ph, H3S10ph or H3S28ph and, therefore, were also filtered from the list.Finally, ratios of filtered marks were log2 transformed and plotted as heat-maps, where histone variants were grouped together for clarity, although these are kept separate in the data tables. […]

Pipeline specifications

Software tools MaxQuant, Andromeda
Application MS-based untargeted proteomics
Chemicals Magnesium