Computational protocol: Phosphoproteomics reveals malaria parasite Protein Kinase G as a signalling hub regulating egress and invasion

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Protocol publication

[…] LC-MS/MS was carried out using an RSLCnano HPLC system (Dionex) and an LTQ-Orbitrap-Velos mass spectrometer (Thermo Scientific). Samples were loaded at high-flow rate onto a reverse-phase trap column (0.3 mm i.d. × 1 mm), containing 5 μm C18 300 Å Acclaim PepMap medium (Dionex) maintained at a temperature of 37 °C. The loading buffer was 0.1% formic acid/0.05% TFA/2% ACN. Peptides were eluted from the column at a flow rate of 0.3 μl min−1 and passed through a reverse-phase PicoFrit capillary column (75 μm i.d. × 400 mm) containing Symmetry C18 100 Å medium (Waters) that was packed in-house using a high-pressure device (Proxeon Biosystems). Peptides were eluted over a period of 4 h, with the output of the column sprayed directly into the nanospray ion source of the LTQ-Orbitrap-Velos mass spectrometer.The LTQ-Orbitrap-Velos mass spectrometer was set to acquire a 1 microscan Fourier transform mass spectrometer (FTMS) scan event at 60,000 resolution over the m/z range 300–2,000 Da in positive ion mode. The maximum injection time for MS was 500 ms and the AGC target setting was 1e6. Accurate calibration of the FTMS scan was achieved using a background ion lock mass for C6H10O14S3 (401.922718 Da). Subsequently, up to ten data-dependent higher-energy collision dissociation (HCD) MS/MS were triggered from the FTMS scan. The isolation width was 2.0 Da, with normalized collision energy of 42.5. Dynamic exclusion was enabled. The maximum injection time for MS/MS was 250 ms and the AGC target setting was 5e4.The raw data file obtained from each LC-MS/MS acquisition was processed using Proteome Discoverer (version 1.4.0.288, Thermo Scientific), searching each file in turn using Mascot (version 2.2.04, Matrix Science Ltd.) against the UniProtKB-Swissprot database. The peptide tolerance was set to 10 p.p.m. and the MS/MS tolerance was set to 0.02 Da. Fixed modifications were set as carboxyamidomethyl (C) and variable modifications set as oxidation (M) and phosphorylation (S/T/Y). A decoy database search was performed.The output from Proteome Discoverer was further processed using Scaffold Q+S (version 4.0.5, Proteome Software). Upon import, the data were searched using X!Tandem (The Global Proteome Machine Organization). PeptideProphet and ProteinProphet (Institute for Systems Biology) probability thresholds of 95% were calculated from the decoy searches and Scaffold was used to calculate an improved 95% peptide and protein probability threshold based on the data from the two different search algorithms. […]

Pipeline specifications

Software tools Proteome Discoverer, Scaffold Q+S, PeptideProphet, ProteinProphet
Application MS-based untargeted proteomics
Organisms Plasmodium falciparum, Homo sapiens
Diseases Malaria
Chemicals Calcium