Project PXD000706

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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 (


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 )

Submission Date


Publication Date



Not available




LTQ Orbitrap Velos



Experiment Type

Shotgun proteomics

Assay count



    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


Showing 1 - 2 of 2 results
# Accession Title Proteins Peptides Unique Peptides Spectra Identified Spectra View in Reactome
1 34470 Control (OTBEH_Gad1Ctrl_Proper2) 293 1242 716 11062 1242
2 34469 PieE (OTBEH_Gad1PieE_proper2) 280 1568 822 10704 1568