Nanolc ultra 2d

Samples were analyzed using an Eksigent NanoLC Ultra 2D (Dublin, CA) and Thermo Fisher Scientific LTQ Orbitrap XL (San Jose, CA). In brief, peptides were first loaded onto a trap cartridge (Agilent), then eluted onto a reversed phase PicoFrit column (New Objective, Woburn, MA) using a linear 120 min gradient of acetonitrile (2–62%) containing 0.1% formic acid at 250 nL/min flowrate. The eluted peptides were sprayed into the LTQ Orbitrap XL. The data-dependent acquisition mode was enabled, and each FTMS MS1 scan (60,000 resolution) was followed by 6 MS2 scans (alternating CID at unit resolution and HCD at 7500 resolution on 3 precursor ions). The spray voltage and ion transfer tube temperature were set at 1.8 kV and 180°C, respectively.

HCT116 cells were transfected with Flag-tagged Oct-1 (Oct-1) or empty vector (Ctrl) for 2 days. Cell lysates were used for IP with Flag-tag antibody-conjugated M2 beads in a buffer containing Tris-buffered saline, 350 mM NaCl and 0.3% NP40. Binding with Protein A/G agarose beads was performed at 4 °C overnight on a rocking platform, followed by six washes in binding solution containing Tris-buffered saline, 350 mM NaCl and 0.3% NP40. After reduction/alkylation (5 mM dithiothreitol, 30 min, 56 °C; and 25 mM iodoacetamide, in the dark, 20 min), 10 ng trypsin and lysC (modified sequencing grade, Roche) in sodium carbonate 50 mM was added and proteins on beads were incubated overnight at 37 °C while shaking. Then the reaction was stopped with 10 μl 10% formic acid. Peptides were recovered and the beads were removed by filtration through C18 Tips (Proxeon) and elution with 20 μl 50% methanol, 5% formic acid, and subjected to LC–MS/MS sequencing using an LC/MS system consisting of an Eksigent NanoLC Ultra 2D (Dublin, CA) and Thermo Fisher Scientific LTQ Orbitrap XL (San Jose, CA). Protein identifications were made using the commercially available search engine Proteome Discoverer 1.4 (Thermo Fisher Scientific).

Excised gel bands were subjected to in-gel trypsin digestion, dehydration with acetonitrile, and rehydration with 50 mM ammonium bicarbonate. Digested peptides were extracted by removing the ammonium bicarbonate solution [28 (link)]. Samples were analyzed using an LC/MS system consisting of an Eksigent NanoLC Ultra 2D (Dublin, CA) and Thermo Fisher Scientific LTQ Orbitrap XL (San Jose, CA). Proteome Discoverer 1.4 (Thermo Fisher Scientific, San Jose, CA) and Sequest algorithms were used for protein identification. Only peptides with a minimum length of six amino acids were considered for identification. Identifications of peptides were also validated by manual inspection of the mass spectra. The LC/MS analysis was performed in the proteomics core facility located at the University of Southern California, Los Angeles, CA.