2.5. Sample preparation for proteomic analysis
ON cells were cultured under proliferative conditions for five days and then lysed (1 x106 cells per condition) in lysis buffer for protein extraction: 1% NP40, 50 mM HEPES [pH 7], 150 mM NaCl, 1 mM EDTA, supplemented with protease inhibitors. Proteins (100 µg/sample) were precipitated in acetone overnight at -20 ºC and recovered by centrifugation at 17,000g for 20 minutes at 4 ºC. Protein pellets were resuspended in 8M Urea in Tris 10 mM [pH 8], reduced with 10 mM dithiothreitol at 50 °C for 30 min and alkylated with 50 mM iodoacetamide for 20 min at room temperature (RT) in the dark. Samples were digested for 4h at RT with Lys-C enzyme/substrate (Promega (V167), USA) (enzyme/substrate ratio 1:50), and then diluted four times with 50 mM ammonium bicarbonate for further trypsin digestion (Promega, USA) at 37 °C overnight (enzyme/substrate ratio 1:50). The digested peptides were desalted using a SepPak C18 cartridge and dried in a SpeedVac, prior to analysis by mass spectrometry (MS) using a label-free quantitative approach. Thus, peptide samples (approximately 500 ng/sample) were loaded onto a nano-ACQUITY UPLC System (Waters, USA) connected on-line to an LTQ Orbitrap XL mass spectrometer (Thermo Electron, USA). An aliquot of each sample was loaded onto a Symmetry 300 C18 UPLC Trap column (180 µm x 20 mm, 5 µm: Waters), and the pre-column was connected to a BEH130 C18 column (75 μm x 200 mm, 1.7 μm: Waters, USA) and equilibrated in 3% acetonitrile and 0.1% formic acid. Peptides were eluted directly into an LTQ Orbitrap XL mass spectrometer (Thermo Finnigan, USA) through a nanoelectrospray capillary source (Proxeon Biosystems, Denmark) at 300 nl/min and using a 120 min linear gradient of 3–50% acetonitrile. The mass spectrometer automatically switched between MS and MS/MS acquisition in data-dependent acquisition mode. Full MS scan survey spectra (m/z 400–2000) were acquired in the orbitrap with mass resolution of 30,000 at m/z 400. After each survey scan, the six most intense ions above 1,000 counts were sequentially subjected to collision-induced dissociation (CID) in the linear ion trap. Precursors with charge states of 2 and 3 were selected specifically for CID and peptides were excluded from further analysis over 60 s using the dynamic exclusion feature.
A Progenesis LC-MS (Waters, USA) apparatus was used for the label-free analysis of differential protein expression. One run was used as a reference, to which the precursor masses in all the other samples were aligned. Only features comprising charges of 2+ and 3+ were selected, and the raw abundances of each feature were normalized automatically and converted to logarithms against the reference run. A peak list containing the information of all the features was generated and exported to the Mascot search engine (Matrix Science Ltd., UK). Differentially expressed proteins were defined as follows: p-value (t-test) <0.05 and fold-change rates > 1.5 for up-regulated or <0.6 for down-regulated proteins identified in at least two replicates. Together with the fold change, the data were finally uploaded into the Ingenuity® Pathway Analysis (IPA) software (Ingenuity Systems, USA) to investigate their molecular and biological functions.