Project description:Synchronized Plasmodium falciparum-infected human blood cultures were harvested at seven time points along the asexual intra-erythrocytic parasite life cycle: 0 hours post-invasion, 6 hpi, 12 hpi, 18 hpi, 24 hpi, 30 hpi and 36 hpi. Plasmodium histones were extracted using the standard hydrochloric acid method and precipitated using trichloroacetic acid. Two biological replicates were digested with three enzymes of diverse specificities: endoproteinase LysC + trypsin (Ti), endoproteinase LysC + GluC (KCEC), and endoproteinase ArgC (RC). The resulting peptide mixtures were analyzed by Multidimensional Protein Identification Technology (MudPIT) coupled to high-resolution tandem mass spectrometry on an LTQ Orbitrap. RAW files were extracted into .ms2 file format using RawDistiller v. 1.0 (in-house developed software). After offline recalibration, the MS/MS spectra were first searched using SEQUEST v.27 (rev.9) with a peptide mass tolerance of 10 ppm and of +/- 0.5 amu for fragment ions. No enzyme specificity was imposed during the SEQUEST search against a protein database combining 5,439 non-redundant Plasmodium falciparum (PlasmoDB release 5.5) and 30,552 human proteins (NCBI 2008-03-04 release, as well as 162 usual contaminants such as human keratins, IgGs and proteolytic enzymes. To estimate false discovery rates (FDR), each protein sequence was randomized (keeping the same amino acid composition and length) and the resulting "shuffled" sequences were added to the database used for the SEQUEST searches, for a total search space of 72,306 amino acid sequences. To account for alkylation by CAM, 57.02146 Da were added statically to cysteine residues for all searches. We set up 44 searches to query for peptides containing various combinations of methylated lysines and arginines (+14.0157), oxidized methionines and hydroxylated lysines, prolines, aspartates, histidines, and tyrosines (+15.9949), formylated lysines and arginines (+27.9949), dimethylated lysines and arginines (+28.0314), trimethylated lysines (+42.0470), acetylated lysines, serines and threonines (+42.0106), crotonylated lysines (+68.0261), phosphorylated serines, threonines, and tyrosines (+79.9663), ubiquitinated lysines (+114.0429), as well as potentially N-terminally acetylated methionines, serines, alanines, and valines (+42.0106), and oxidized methionines (+58.0055). The maximum number of modified amino acids per differential modification in a peptide was limited to six. The MS/MS dataset was also searched using the same parameters with OMSSA to validate newly described PTMs.

Project description:Sample preparation, protein digestion, and MS data acquisition are described in MSV000086790. Post-translational Modification Searches | Peak files were extracted using RawDistiller and searched using ProLuCID (v. 1.3.3) against a database consisting of 5,846 S. cerevisiae non-redundant proteins (downloaded from NCBI 11-01-2016), 193 usual contaminants (such as human keratins, IgGs, and proteolytic enzymes), and, to estimate false discovery rates (FDRs), 6039 randomized amino acid sequences derived from each non-redundant protein entry. All cysteines were considered as fully carboxamidomethylated (+57.0215 Da statically added). The MS/MS dataset for the samples only digested with trypsin was further searched for modified peptides in a recursive manner. A total of eight differential modification searches were set up to query for oxidized methionines (+15.9949 Da) in combination with: 1) acetylated lysines, serines, and threonines (+42.0106 Da); 2) formylated arginines and lysines (+27.9949 Da); 3) hydroxylated aspartates, tyrosines, histidines, and prolines (+15.9949); 4) mono- (+14.0157 Da) and 5) dimethylated (+28.0314 Da) arginines and lysines; 6) trimethylated (+42.0470) lysines; 7) phosphorylated (+79.9663 Da) serines, threonines, and tyrosines; and 8) ubiquitinated lysines (+114.0429 Da). Mass tolerances of 7 ppm for precursor and 800 ppm for fragment ions were used. MS/MS datasets generated for trypsin-digested peptides were searched with KR peptide-end requirements. After this round of searches, an in-house software, sqt-merge (Zybailov et al 2005), was used to combine the ProLuCID output files (.sqt files) allowing only the best matches out of the eight differential queries to be ranked first based on cross-correlation scores (XCorr) and for normalized differences between XCorrs (DeltaCn) to be recalculated accordingly. DTASelect v1.9 and swallow v. 0.0.1 (https://github.com/tzw-wen/kite) were used to filter ProLuCID search results at given FDRs at the spectrum, peptide, and protein levels. Here, all controlled FDRs were less than 1%.

Dataset Information

Histone modifications in Plasmodium falciparum at 36 hours post-invasion

Dataset's files

Publications

Dynamic and Combinatorial Landscape of Histone Modifications during the Intraerythrocytic Developmental Cycle of the Malaria Parasite.

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