Computational protocol: Role of the Plasmodium Export Element in Trafficking Parasite Proteins to the Infected Erythrocyte

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Protocol publication

[…] 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). The parameters used to generate the peak lists for the Orbitrap were as follows: minimum mass 400, maximum mass 5000, grouping tolerance 0.01 Da, intermediate scans 1, minimum group count 1 and 10 peaks minimum and total ion current of 100. Peak lists for each nano-LC-MS/MS run were used to search MASCOT v2.2.04 search algorithm (Matrix Science) provided by the Australian Proteomics Computational Facility (www.apcf.edu.au). Automatic charge state recognition was used because of the high-resolution survey scan (30 000). LC-MS/MS files were searched against a human, mouse, bovine and P. falciparum database containing the PEXEL constructs and also including a small contaminant database (keratins, trypsin, chymotrypsin, etc.). The search parameters consisted of carboxymethylation of cysteine as a fixed modification (+58 Da) with variable modifications set for NH2-terminal acetylation (+42 Da) and oxidation of methionine (+16 Da). A peptide mass tolerance of ±20 p.p.m., #13C defined as 1, fragment ion mass tolerance of ±0.8 Da and an allowance for up to three missed tryptic cleavages for Orbitrap data.The signal sequence processing site of KAHRP was predicted by SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP/) with both neural networks and hidden Markov models trained on eukaryotes . The signal sequence processing site of GBP130 was predicted as above with SignalP 1.1 (http://www.cbs.dtu.dk/services/SignalP-1.1/) . […]

Pipeline specifications

Software tools Mascot Server, SignalP
Application MS-based untargeted proteomics
Organisms Plasmodium falciparum, Homo sapiens