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Interactome of the Legionella pneumophila effector PieE
The in vivo interactome of the Legionella pneumophila effector PieE reveals binding to multiple Rab GTPases
Sample Processing Protocol
For the LC-MS/MS analysis, the proteins on the streptavidin resin were reduced with 400uL of 5mM Tris(2-carboxyethyl)phosphine hydrochloride (TCEP, Sigma) and alkylated with 10mM Iodoacetamide (IAA, Sigma). 1.5 µg Lys-C (Roche) was added and incubated overnight at 25°C, then 2.0 µg trypsin (Promega) was added to further digest for 6 h at 35°C, and the supernatant was collected. Formic acid (FA) was added to the supernatant to a final concentration at 1% and 50µL of 25% acetonitrile/0.1% FA to further extract peptides on the beads. The two eluted solutions were pooled and dried down in a SpeedVac (Thermo). The peptides were reconstituted with 0.1% FA/H2O prior to mass spectrometric analysis. The samples were analysed with on-line nanoLC-MS/MS on an Ultimate 3000 Capillary/nano HPLC System (Dionex) coupled to a LTQ Orbitrap Velos hybrid mass spectrometer (Thermo Fisher) equipped with a nanospray source. Samples were first loaded and desalted on a PepMap C18 trap (0.3 mm id x 5 mm, 5µm, Dionex) then peptides were separated on a 75 µm id x 10 cm BEH C18 column (1.7 µm, Waters) over a 60 min linear gradient of 4–32% CH3CN/0.1% FA at a flow rate at 300nL/min. The LTQ Orbitrap Velos was operated in the “Top 10” CID data-dependant acquisition mode where MS survey scan in the Orbitrap was m/z 380 – 160 with the lock mass at 445.120025 at resolution 60,000 at m/z 400.
Data Processing Protocol
The raw files were processed with Proteome Discoverer 1.3 using Mascot search engine (v2.3, Matrix Science) with the following parameters: trypsin/P with maximum 2 missed cleavages sites; peptide mass tolerance at first search was set at 10 ppm; MS/MS fragment mass tolerance at 0.49 Da. Fixed modification for Carbamidomethyl and variable modifications for Acetyl (Protein N-term), Deamidated (NQ), and Oxidation (M) were used. The protein databases were combined with human IPI, E.coli and Legionella pneumophila 130b. The Mascot result files were further processed with in-house Mascot Percolator (http://www.sanger.ac.uk/resources/software/mascotpercolator/).
James Wright, Functional Proteomics, Institute Cancer Research
Proteomic Mass Spectrometry, Wellcome Trust Sanger Institute
Jyoti Choudhary, Proteomics Mass Spectrometry, Wellcome Trust Sanger Institute ( lab head )
Mousnier A, Schroeder GN, Stoneham CA, So EC, Garnett JA, Yu L, Matthews SJ, Choudhary JS, Hartland EL, Frankel G. A new method to determine in vivo interactomes reveals binding of the Legionella pneumophila effector PieE to multiple rab GTPases. MBio. 2014 Aug 12;5(4). pii: e01148-14 PubMed: 25118235