and R

and R.P.G. interest in developing novel therapeutic strategies in the treatment and management of pancreatic cancer. Patient-derived tumor xenografts (PDXs) in mice serve as potentially valuable preclinical models as they maintain the histological and molecular heterogeneity of the original human tumor. Here, we employed high-resolution mass spectrometry combined with immunoaffinity purification using anti-phosphotyrosine antibodies to profile tyrosine phosphoproteome across 13 pancreatic ductal adenocarcinoma PDX models. This analysis resulted in the identification of 1199 tyrosine-phosphorylated sites mapping to 704 proteins. The mass spectrometric analysis revealed widespread and heterogeneous activation of both receptor and non-receptor tyrosine kinases. Preclinical studies confirmed ephrin type-B receptor 4 (EphB4) as a potential therapeutic target based on the efficacy of human serum albumin-conjugated soluble EphB4 in mice bearing orthotopic xenografts. Immunohistochemistry-based validation using tissue microarrays from 346 patients with PDAC showed significant expression of EphB4 in 70% of patients. In summary, we present a comprehensive landscape of Diazepam-Binding Inhibitor Fragment, human tyrosine phosphoproteome with EphB4 Diazepam-Binding Inhibitor Fragment, human as a promising Diazepam-Binding Inhibitor Fragment, human therapeutic target in pancreatic ductal adenocarcinoma. at 15 C for 15 min. Protein samples of 25 mg from each were reduced with 5 mM dithiothreitol for 60 min at 37 C and alkylated with 10 mM iodoacetamide for 30 min in the dark at room temperature. Samples were Sema3a diluted with 20 mM HEPES pH 8.0 to a final concentration of 2 M urea and incubated with TPCK-treated trypsin at room temperature overnight with gentle end-to-end shaking. Protein digests were acidified by adding 20% trifluoroacetic acid (TFA) to a final concentration of 1% TFA and subjected to centrifugation at 2000 at room temperature for 15 min. The supernatant of protein digests was loaded onto a Sep-Pak C18 cartridge (Waters Corporation, Milford, MA, USA) pre-equilibrated Diazepam-Binding Inhibitor Fragment, human with 0.1% TFA. Peptides were eluted with 40% acetonitrile with 0.1% TFA. Eluted peptides were lyophilized and subjected to immunoaffinity purification of tyrosine-phosphorylated peptides. 2.4. Immunoaffinity Purification of Tyrosine Phosphopeptides Immunoaffinity purification (IAP) of tyrosine-phosphorylated peptides was carried out as previously described [11]. Briefly, after lyophilization, about 25 mg of tryptic peptides was dissolved in 1.4 mL of 1 1 IAP buffer (50 mM MOPS pH 7.2, 10 mM sodium phosphate, 50 mM NaCl) and subjected to centrifugation at 2000 at room temperature for 5 min. Before immunoaffinity purification, P-Tyr-1000 beads were washed with 1 IAP buffer twice at 4 C, and the pH of the supernatant containing peptides was adjusted to ~7.2 by adding 1 M Tris Base. For immunoaffinity purification, the supernatant was incubated with P-Tyr-1000 beads at 4 C for 60 min, and the beads were washed three times with 1 IAP buffer and twice with cold ultrahigh pure water. Peptides captured by antibody were eluted twice by incubating the beads with 0.15% TFA at room temperature for 15 min. Eluted peptides were desalted by C18 STAGE tip, vacuum dried, and kept at ?80 C prior to LC-MS/MS experiments. 2.5. Liquid Chromatography-Tandem Mass Spectrometry LC-MS/MS analysis of the enriched tyrosine-phosphorylated peptides was carried out using a reversed-phase liquid chromatography system (Ultimate 3000 RSLCnano, Thermo Scientific, San Jose, CA, USA) connected online to an Orbitrap Fusion Lumos mass spectrometer (Thermo Scientific, San Jose, CA, USA). The peptides were loaded onto a trap column (PepMap C18 2 cm 100 m, and 100 ?) at a flow rate of 20 L/min using 0.1% formic acid Diazepam-Binding Inhibitor Fragment, human and separated on an analytical.