br Dehydrated samples were suspended in L of
Dehydrated samples were suspended in 20 µL of NuPAGE LDS sample buffer (ThermoFisher Scientific). SILAC pairs were mixed, denatured at 90 °C for 2 min and separated on a 10% NuPAGE gel (ThermoFisher Scientific) at 120 V for 20 min. The gel was washed and silver stained (Pierce Silver Stain kit). Each lane was excised and destained using 30 mM potassium hexa-cyanoferrate (III) /100 mM sodium thiosulfate, washed and de-hydrated using a vacuum centrifuge. Proteins in the gel slabs were reduced, alkylated with iodoacetamide, dehydrated in a vacuum centrifuge. Gel slabs
were then re-hydrated using 50 mM ammonium bicarbonate pH 8.4 (containing 0.04 µg sequencing grade modified porcine trypsin (Promega)) and incubated for 16 h at 37 ○C. After proteolysis inhibition and tryptic peptide elution from the polyacrylamide slabs, eluates from each lane were pooled, lyophilized, and stored at −80 ○C until analysis. The lyophilized peptide pellets were resuspended in 0.1% formic acid/ 3% acetonitrile and 5% of the solution was analyzed by LC/MS. The LC system consisted of a vented trap-elute setup (EasynLC1000, Thermo Fisher scientific) coupled to the Orbitrap Fusion mass spectrometer (Thermo Fisher Scientific, San Jose, CA) via a nano electro-spray DPV-565 PicoView ion source (New Objective). The trap column was fabricated with a 5 cm × 150 µm internal diameter silica capillary with a 2 mm silicate frit, and pressure loaded with Poros R2-C18 10 µm particles (Life Technologies). The analytical column consisted of a 25 cm × 75 µm internal diameter column with an integrated electrospray emitter (New Objective), packed with ReproSil-Pur C18-AQ 1.9 µm particles (Dr. Maisch). Peptides were resolved over 90 min using a 3%–45% gradient acetonitrile/ 0.1% formic Herboxidiene (buffer
automatic gain control target set at 105 ions and a maximum injection time of 50 ms. We used data-dependent mass spectral acquisition with monoisotopic precursor selection, ion charge selection (2–7), dynamic precursor exclusion (60 s, 20 ppm tolerance) and HCD fragmentation (normalized collision energy 35, isolation window 0.8 Th) using the top speed algorithm with a duty cycle of 2 s. Product ion spectra were recorded in the linear ion trap (“normal” scan rate, automatic gain control = 5000 ions, maximum injection time = 150 ms). Spectra were analyzed using MaxQuant Version188.8.131.52, searching within the human UniProt database (version 01/27-2016) with FDR <0.01. Intensity measurements were normalized to the PP2A-Aα intensity and then SILAC ratios were calculated. Four biological replicates were analyzed. P-values were calculated from a two-tailed student’s t-test for proteins identified in three or more experiments. We focused on proteins with > 2-fold change in binding and P < 0.05. Raw files are openly accessible via PRIDE accession number PXD010709.
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Expert Systems With Applications
journal homepage: www.elsevier.com/locate/eswa
A cascade-learning approach for automated segmentation of tumour epithelium in colorectal cancer
Mohammed M. Abdelsamea a,b,∗, Alain Pitiot c, Ruta Barbora Grineviciute d,e, Justinas Besusparis d,e, Arvydas Laurinavicius d,e, Mohammad Ilyas f