Computational protocol: Identification of multiple roles for histone acetyltransferase 1 in replication-coupled chromatin assembly

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Protocol publication

[…] RAW data were uploaded to MaxQuant (v1.5.3.17) () and searched against a murine UniProt Swiss-Prot database (downloaded 20 November 2015; 16 741 entries) using the Andromeda search engine. Three biological replicates were analyzed as separate experiments, each with two combined technical analyses. Oxidation of M and acetylation of protein NH2-termini were included as variable modifications and carbamidomethylation of C was included as a fixed modification. Up to two missed cleavages were allowed and the peptide minimum length was set to seven residues with up to three amino acids labeled with Arg10/Lys8. Enzyme specificity was set for trypsin/P. The peptide mass tolerance was fixed at 20 ppm with a fragment mass tolerance of 0.5 Da. Protein and PSM FDRs were set at 0.05. The match between runs feature was enabled with default settings. Quantitation was performed for proteins with at least two unique or razor peptides and any of the variable/fixed modifications described above.Heavy/light SILAC ratios were calculated in Perseus using intensity values from the MaxQuant proteinGroups output file. Proteins with three valid SILAC ratios (i.e. proteins quantified on both channels in at least one of two technical replicates across all three biological replicates) were kept for further analysis. A two-tailed t-test was performed in Excel followed by the calculation of a multi-test corrected q-value. Data were visualized in Excel or R. […]

Pipeline specifications

Software tools MaxQuant, Perseus
Databases UniProt
Application MS-based untargeted proteomics
Organisms Mus musculus, Caenorhabditis elegans