Computational protocol: PRAS40 and PRR5-Like Protein Are New mTOR Interactors that Regulate Apoptosis

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Protocol publication

[…] The peptides were analyzed by two-dimensional capillary liquid chromatography and tandem MS using a PolySULFOETHYL A ion-exchange column (0.15×50 mm, PolyLC, Columbia, MD), connected in series to a C18 trap column (Zorbax 300SB, 0.3×50 mm, Agilent Technologies, Basel, Switzerland), and to a Magic C18 separation column (0.1×100 mm, Thermo Scientific, Basel, Switzerland). The peptides were injected first onto the cation exchange column. Unadsorbed peptides were trapped on the Zorbax column and eluted onto the separation column with a linear 75 min gradient from 2 to 75% B (0.1% acetic acid in 80% acetonitrile) in solvent A (0.1% acetic acid in 2% acetonitrile). Next, peptides that had been retained by the ion-exchange column were sequentially eluted and trapped on the C18 trap column with 10 mL pulses of 50, 100, 150, 200, 250, 300, 350, 400, and 500 mM ammonium acetate, pH 3.3. Peptides eluted by each individual salt pulse were separated by the acetonitrile gradient as described above. The flow was delivered with a Rheos 2200 HPLC system (Thermo Scientific, Basel, Switzerland) at 50 mL/min. A precolumn splitter reduced the flow to approximately 500 nl/min. The eluting peptides were ionized by a Finnigan nanospray ionization source (Thermo Scientific, Basel, Switzerland). The LTQ orbitrap instrument was operated in the data-dependent mode. A survey scan was performed in the Orbitrap between m/z 400-1600 Da at 60,000 resolution. The three most abundant ions detected were fragmented in the LTQ mass spectrometer and mass analyzed in the Orbitrap at a resolution of 7,500. Singly charged ions were not subjected to fragmentation. The normalized collision energy was set to 35%. Individual MS/MS spectra were searched against the NCBI non-redundant databank using the TurboSequest software . The Sequest filter parameters were as follows: Xcorr versus charge state was 1.50 for singly, 2.00 for doubly, and 2.50 for triply charged ions, respectively; the ΔCN was 0.1, and the protein probability was set to 0.01. […]

Pipeline specifications

Software tools EMBOSS, Comet
Application MS-based untargeted proteomics
Organisms Pseudomonas phage PRR1, Homo sapiens
Diseases Tuberous Sclerosis
Chemicals Cycloheximide, Sirolimus