4.11. Label-Free Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS)

The discovery phase (shotgun proteomics) was performed in two subcellular fractions (nucleus and cytoplasm) from two SCCC cell lines (C-33A and SiHa) with three biological replicates for each experimental condition: MOCK, NC-transfected and anti-miR-877-3p-transfected cells (36 samples in total). The size of the groups meant that the biological variation could be evened out. Protein extracts for each sample were diluted in Laemmli sample buffer and loaded into a 0.75-mm-thick polyacrylamide gel with a 4% stacking gel cast on a 12.5% resolving gel. The run was stopped as soon as the front had penetrated 3 mm into the resolving gel so that the whole proteome became concentrated in the stacking/resolving gel interface. Bands were stained with Coomassie Brilliant Blue and excised from the gel. Protein enzymatic cleavage (20 µg) was carried out with trypsin (1:20, w/w) (Promega, Madison, WI, USA) at 37 °C for 16 h, as previously described [111]. Peptides were purified and concentrated using C18 Zip Tip Solid Phase Extraction (Millipore, Burlington, MA, USA). Peptide mixtures were separated by reverse-phase chromatography using an Eksigent nanoLC ultra 2D pump fitted with a 75-μm ID column (Eksigent 0.075 × 250). Samples were first loaded for desalting and concentration into a 0.5-cm length 100-μm ID precolumn, packed with the same chemicals as the separating column. Mobile phases were 100% water with 0.1% formic acid (FA) (buffer A), and 100% acetonitrile with 0.1% FA (buffer B). The column gradient was developed in a 240-min two-step gradient from 5% B to 25% B in 210 min and 25% B to 40% B in 30 min. The column was equilibrated in 95% B for 9 min and 5% B for 14 min. Throughout the process, the precolumn was in line with the column and the flow was maintained along the gradient at 300 nl/min. Eluting peptides from the column were analyzed with a Sciex 5600 Triple-TOF system (Sciex, Framingham, MA, USA). Data were acquired from a survey scan performed on a mass ranging from 350 to 1250 m/z in a scan time of 250 ms. The top 35 peaks were selected for fragmentation. The minimum accumulation time for MS/MS was set to 100 ms, giving a total cycle time of 3.8 s. Product ions were scanned in a mass range from 230 to 1500 m/z and were excluded from further fragmentation for 15 s.