Computational protocol: CRKL mediates EML4-ALK signaling and is a potential therapeutic target for ALK-rearranged lung adenocarcinoma

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[…] H3222 and H2228 cells (1×108) were treated with DMSO (as control) or crizotinib (10nM, 100nM and 1000nM) for 3hrs. Phosphopeptides were purified by using Cell Signaling PhosphoScan pTyr100 Kits (Beverly, MA) according to the manufacturer's recommendations. Briefly, cells were lysed in urea lysis buffer (20mM HEPES pH 8.0, 9M urea, 1mM sodium orthovanadate, 2.5 mM sodium pyrophosphate, 1mM β-glycerophosphate) at 1x10^7 cells/ml and sonicated. Lysates were cleared by centrifugation at 20,000 x g for 15 minutes, and proteins were reduced by 4.5mM of dithiothreitol and alkylated by 10mM of iodoacetamide. Samples were diluted with 200 mM HEPES pH 8.0 to a final concentration of 2M urea and 20mM HEPES. Trypsin (TPCK-treated; 1mg/ml) was then added to the lysate at 1:100 (v/v) ratio. Samples were digested overnight at room temperature with gentle shaking. Following digestion, lysates were acidified to a final concentration of 1% trifluoroacetic acid (TFA). Peptide purification was carried out using Sep-Pak C18 columns. All elutions (10%, 15%, 20%, 25%, 35% and 40% acetonitrile in 0.1% TFA) were combined into one fraction and lyophilized. Dried peptides were resuspended in 1.4ml of MOPS IP buffer (50mM MOPS pH7.2, 10mM Na3PO4, 50mM NaCl), and insoluble materials were removed by centrifugation at 2000 x g for 5 minutes. Solubilized peptides were transferred into the microcentrifuge tube containing phosphotyrosine p-Tyr-100 antibody beads (40μl slurry for each sample) and the mixture was incubated overnight at 4°C. The immobilized antibody beads were washed three times with 1ml MOPS IP buffer and twice with 1ml water (ice-cold). Peptides were eluted from beads by incubation with 55 μl of 0.1% TFA, followed by a second elution with 45 μl of 0.1% TFA, and both fractions were combined. [...] The IP-enriched peptides were analyzed on an LTQ Orbitrap Velos (Thermo Fisher Scientific, San Jose, CA) coupled with a nanoLC system (Eksigent nanoLC-Ultra 1D plus, Dublin, CA). Peptides were separated on a C18 reversed phase column (100mm long, ID 75μm, 5μm 300Å BetaBasic, New Objective, Woburn, MA) using a 90-min linear gradient from 2% to 35% acetonitrile in 0.1% formic acid at a flow rate of 250nL/min. Mass analysis was conducted in data-dependent analysis (DDA) mode, where MS1 scanned mass range from 300 to 2000 m/z at 30,000 resolution in the Orbitrap, and 10 collision-induced dissociation (CID) MS2 scans were sequentially carried out in the ion trap.The LC-MS data were searched against the SwissProt Human database using Mascot server (Matrix Science, London, UK; version 2.3). Searching parameters were set as: precursor mass tolerance at 20ppm, fragment ion mass tolerance at 0.8 Da, trypsin enzyme with 2 miscleavages, carbamidomethylation of cysteine as fixed modification, and deamidation of asparagine and glutamine, oxidation of methionine, and tyrosine/serine/threonine phosphorylation as variable modifications. Peptides identified from database search were filtered with a false discovery rate (FDR) of 0.05. Relative quantitation of phosphopeptides was calculated based on areas under the curve (AUC) of corresponding peptides from the control and treated samples. […]

Pipeline specifications

Software tools PhosphoScan, Mascot Server
Application MS-based untargeted proteomics
Chemicals Tyrosine