Dataset Information

Comparative phosphoproteome of Trypanosoma brucei bloodstream & procyclic form

ABSTRACT: Comparative phosphoproteomic analysis of SILAC labelled cultured bloodstream and procyclic form Trypansoma brucei. Phosphopeptide enrichement via SCX and TiO2, acquired on Oribtrap Velos using MSA. Triplicate biological replicate for each lifecycle stage (as unmixed, and two SILAC label swap expreiments), eight fraction injected in technical triplicate. For the SILAC labelled samples, the corresponding changes in the proteome were measured using unenriched peptides sperated by SCX. Data was processed using MaxQuant version 1.3.0.5 which incorporates the Andromeda search engine. Proteins were identified by searching a protein sequence database containingT. brucei brucei 927 annotated proteins (Version 4.0, downloaded from TriTrypDB http://www.tritrypdb.org/ supplemented with the VSG221 sequence and frequently observed contaminants (porcine trypsin, bovine serum albumins and mammalian keratins) that contains a total of 10,081 protein sequences. Search parameters specified an MS tolerance of 6 ppm, an MS/MS tolerance at 0.5 Da and full trypsin specificity, allowing for up to two missed cleavages. Carbamidomethylation of cysteine was set as a fixed modification and oxidation of methionines, N-terminal protein acetylation and N-pyroglutamate were allowed as variable modifications. Phosphoproteomic analysis included phosophorylation of serine, threonine and tyrosine as additional variable modifications. Peptides were required to be at least 7 amino acids in length and a MaxQuant score >5, with false discovery rates (FDRs) of 0.01 calculated at the levels of peptides, proteins and modification sites based on the number of hits against the reversed sequence database.

INSTRUMENT(S): LTQ Orbitrap Velos

ORGANISM(S): Trypanosoma Brucei

SUBMITTER: Michael Urbaniak

LAB HEAD: Michael Urbaniak

PROVIDER: PXD000049 | Pride | 2013-07-03

REPOSITORIES: Pride

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Global quantitative SILAC phosphoproteomics reveals differential phosphorylation is widespread between the procyclic and bloodstream form lifecycle stages of Trypanosoma brucei.

Urbaniak Michael D MD Martin David M A DM Ferguson Michael A J MA

Journal of proteome research 20130329 5

We report a global quantitative phosphoproteomic study of bloodstream and procyclic form Trypanosoma brucei using SILAC labeling of each lifecycle stage. Phosphopeptide enrichment by SCX and TiO2 led to the identification of a total of 10096 phosphorylation sites on 2551 protein groups and quantified the ratios of 8275 phosphorylation sites between the two lifecycle stages. More than 9300 of these sites (92%) have not previously been reported. Model-based gene enrichment analysis identified over ...[more]

PMID: 23485197

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Project description:Wild type yeast cells were metabolically labeled in SILAC medium. The heavy (H)-SILAC-labeled cells were then continuously exposed to 0.01% MMS to induce replication stress, and the light (L)-SILAC-labeled cells were mock-exposed. After 3 hours, heavy and light cells were harvested and lysed. To ensure reproducibility, the entire experiment was repeated, and the labels were swapped such that the (L)-SILAC-labeled wild type yeast cells were exposed to 0.01% MMS for 3 hours, and the (H)-SILAC-labeled wild type yeast cells were mock-exposed. Lysates from heavy and light cells were mixed 1:1 by protein mass, reduced and treated with iodoacetamide and then digested with trypsin. 5 mg of each protein lysate was fractionated by high-pH reverse phase (RP) liquid chromatography into 12 samples. For proteome analysis, 2% of each fraction was dried down and re-suspended for LC-MS/MS analysis. The remaining 98% was processed to enrich for phosphopeptides using immobilized metal affinity chromatography (IMAC), dried down, and re-suspended in 0.1% FA, 3% ACN for LC-MS/MS analysis. Global and phosphopeptide-enriched samples were analyzed by LC-MS/MS on a Thermo LTQ-Orbitrap Velos mass spectrometer. Raw MS/MS spectra were searched against version 3.69 of the Yeast International Protein Index (IPI) sequence database using three independent search engines (MaxQuant/Andromeda, Spectrum Mill, and xTandem). All searches were performed with the tryptic enzyme constraint set for up to two missed cleavages, oxidized methionine set as a variable modification, and carbamidomethylated cysteine set as a static modification. For MaxQuant, the peptide MH+ mass tolerances were set at 20 ppm. For X!Tandem, the peptide MH+ mass tolerances were set at ±2.0 Da with post-search filtering of the precursor mass to 50 ppm and the fragment MH+ mass tolerances were set at ±0.5 Da. For Spectrum Mill, peptide MH+ mass tolerances were set at 20 ppm and fragment MH+ mass tolerances were set at ±0.7 Da. The overall FDR was set at ≤3% based on a decoy database search. Phosphosite localization probabilities are reported in the MaxQuant results. Any site with a probability greater than 0.8 was considered to be localized. Quantification of the Heavy:Light ratios was performed using MaxQuant software, with a minimum ratio count of 2 and using unique + razor peptides for quantification.

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