Computational protocol: The B-cell antigen receptor signals through a preformed transducer module of SLP65 and CIN85

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[…] Proteins were separated by one-dimensional SDS–PAGE (4–12% NuPAGE Bis-Tris Gel, Invitrogen) and the entire lane of the Coomassie blue-stained gel was cut into 23 slices. All slices were reduced with 10 mM DTT for 55 min at 56°C, alkylated with 55 mM IAA for 20 min at 26°C and digested with modified trypsin (Promega) overnight at 37°C. Tryptic peptides were injected into a C18 precolumn (1.5 cm, 360 μm o.d., 150 μm i.d., Reprosil-Pur 120 Å, 5 μm, C18-AQ, Dr Maisch GmbH) at a flow rate of 10 μl/min. Bound peptides were eluted and separated on a C18 capillary column (15 cm, 360 μm o.d., 75 μm i.d., Reprosil-Pur 120 Å, 5 μm, C18-AQ, Dr Maisch GmbH) at a flow rate of 300 nl/min, with a gradient from 7.5 to 37.5% ACN in 0.1% formic acid for 60 min using an Agilent 1100 nano-flow LC system (Agilent Technologies) coupled to an LTQ-Orbitrap XL hybrid mass spectrometer (Thermo Electron). MS conditions were as followed: spray voltage, 1.8 kV; heated capillary temperature, 150°C; normalized collision-induced dissociation (CID) collision energy 37.5% for MS/MS in LTQ. An activation q=0.25 and activation time of 30 ms were used. The mass spectrometer was operated in the data-dependent mode to automatically switch between MS and MS/MS acquisition. Survey MS spectra were acquired in the Orbitrap (m/z 350–1600) with the resolution set to 30 000 at m/z 400 and automatic gain control target at 5 × 105. The five most intense ions were sequentially isolated for CID MS/MS fragmentation and detection in the linear ion trap. Ions with single and unrecognized charge states were excluded. Raw data were analysed with MaxQuant software (Version 1.0.12.31) in combination with Mascot search engine for peptide and protein identifications (Version 2.2.04, Matrix Science). IPI Chicken (Version 3.47) was used as Gallus gallus sequence database. MS/MS peak lists were filtered to contain at most six peaks per 100 Da interval and searched against Mascot server. The MS mass tolerance was set to 7 p.p.m. and MS/MS mass tolerance was set to 0.8 Da. Up to three missed cleavages of trypsin were allowed. Oxidized methionine and cysteine carbamido-methylation were searched as variable modifications. The modifications corresponding to arginine and lysine labelled with heavy stable isotopes were handled as fixed modifications in the Mascot search, if applicable, after identification of SILAC pairs by MaxQuant. The false positive rate was set to 1% at the peptide level, the false discovery rate was set to 1% at the protein level and the minimum required peptide length was set to six amino acids. […]

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