Computational protocol: Mapping the interactome of overexpressed RAF kinase inhibitor protein in a gastric cancer cell line

Similar protocols

Protocol publication

[…] IPGphor, Ettan DALT II System, Image-Scanner (maximum resolution 9,600 × 9,600 dpi; Amersham Biosciences, Stockholm, Sweden), electrospray ionization-quadrupole-time of flight (ESI-Q-TOF) Mass Spectrometer (Micromass, Manchester, UK), PDQuest system (Bio-Rad laboratories, Hercules, CA), Mascot Distiller and Mascot Database Search engine, and Statistical Package For Social Science (SPSS for windows, Version 17.0, Chicago, IL, USA) were used. [...] After 1D SDS-PAGE separation of the purified proteins from three groups (RKIP-3xFLAG, 3xFLAG, and Blank), respectively,the proteins that were contained in the gel bands were digested with trypsin, and the tryptic peptide mixture was analyzed with Micromass ESI-Q-TOF MS/MS. The tryptic peptide samples were loaded onto a pre-column (320 μm × 50 mm, 5 μm C18 silica beads; Waters) to be concentrated and quickly desalted (30 l/min flow-rate) through a Waters CapLC autosampler. The concentrated and desalted tryptic peptides were online eluted to the reversed-phase column (75 μm × 150 mm, 5 mm, 100 Å, LC Packing) at a flow-rate of 200 nl/min. MS/MS spectra were acquired in a data-dependent mode in which up to four precursor ions above an intensity threshold of seven counts/second (cps) from each survey “scan” were selected for MS/MS analysis. The nanospray parameters were the following: a 3,000 V capillary voltage, a 45 V cone voltage, an 80°C source temperature, and a 15-psi collision gas back pressure. The MS/MS data were used to search the identified proteins against the protein database with the Mascot search engine. The search parameters used were the following: homo sapiens as the current species, a mass tolerance of +0.5 Da, an MS/MS tolerance of +0.3 Da, up to 1 missed cleavage site, fixed carboxymethyl (cysteine) modification, variable oxidation (methylation) modification, the Micromass PKL format, and the ESI Q-TOF instrument. The proteins identified from the blank-carrier control group and from the blank control group were regarded as non-specific proteins, and were removed from the protein list identified from the pcDNA3.1-RKIP-3xFLAG test group to rule out the non-specifically binding proteins of RKIP. […]

Pipeline specifications

Software tools PDQuest 2-D, Mascot Distiller, Mascot Server
Application MS-based untargeted proteomics
Organisms Homo sapiens
Diseases Multiple Sclerosis, Stomach Neoplasms