MudPIT PTMs analyses of whole cell protein extracts from Saccharomyces cerevisiae grown at 21C and 35C
Ontology highlight
ABSTRACT:
INSTRUMENT(S): LTQ Orbitrap Velos
ORGANISM(S): Saccharomyces Cerevisiae (ncbitaxon:4932)
SUBMITTER: 
Laurence Florens
PROVIDER: MSV000086791 | MassIVE | Mon Feb 01 17:19:00 GMT 2021
SECONDARY ACCESSION(S): PXD023925
REPOSITORIES: MassIVE
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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.
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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.
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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.
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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.
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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.
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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.
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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.
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