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Protocol publication

[…] For protein identification of protein spots LC-MS/MS data were searched against a non-redundant protein decoy database comprising sequences from P. falciparum, as well as their reverse sequences and common contaminants. Mass spectra peak lists were extracted using extract-msn as part of Bioworks 3.3.1 (Thermo Fisher Scientific) linked into Mascot Daemon (Matrix Science, UK). Peak lists for each nano-LC-MS/MS run were searched using MASCOT v2.2.04 (Matrix Science, UK), provided by the Australian Proteomics Computational Facility (www.apcf.edu.au). The search parameters consisted of carbamidomethylation of cysteine as a fixed modification (+57 Da), with variable modifications set for NH2-terminal acetylation (+42 Da) and oxidation of methionine (+16 Da). A precursor mass tolerance of 20 ppm, fragment ion mass tolerance of ±0.04 Da and an allowance for up to three missed cleavages for tryptic searches was used. Scaffold v4.4.3 (Proteome Software, Inc., USA) was used to validate protein identifications derived from MS/MS sequencing results (FDR <0.5%). [...] Relative quantitation of differentially labeled RBC phosphopeptide preparations was performed using the MaxQuant software package (). High-resolution MS data were searched using a tolerance of 10 ppm for precursor ions and 20 mmu for productions. Enzyme specificity was tryptic and allowed for up to two missed cleavages per peptide. Carbamidomethylation of cysteines (+57) was specified as a constant modification with oxidation of methionine (+16), protein N-terminal acetylation (+52) and light (L; △ = +28), intermediate (M; △ = +32) or heavy (H; △ = +36) dimethyl labels on the peptide N termini and lysine residues set as variable modifications. The MS data were searched against human, bovine, or P. falciparum proteins in the non-redundant MSPnr protein database at a 1% false discovery rate (FDR). A robust permutation test was used to analyze MaxQuant data and evaluate statistically significant differences in the relative abundance of RBC phosphopeptides (). […]

Pipeline specifications

Software tools Mascot Server, MaxQuant
Application MS-based untargeted proteomics
Organisms Homo sapiens, Toxoplasma gondii
Diseases Malaria, Musculoskeletal Diseases, Malaria, Falciparum