JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig2_TOP06_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig2_TOP04_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_C_03.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_B_03.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_D_03.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_A_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_E_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_C_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_B_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_E_03.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig2_TOP12_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_D_01.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig2_TOP06_02.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_C_02.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_A_02.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_B_02.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_D_02.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_A_03.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig2_TOP04_02.rawhttps://storage.jpostdb.org/JPST000075/files/HCT2mg_fig1_E_02.rawprimaryOK200ProteomicsTakeshi TomonagaHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST000075Gifu UniversityjPOSTPhosphorylation is a major post-translational modification that regulates protein function, with phosphotyrosine (pY) modifications being implicated in oncogenesis. However, global profiling of pY statuses without treatment with a tyrosine phosphatase inhibitor such as pervanadate is still challenging due to the low occupancy of pY sites. In this study, we greatly improved the identification of pY sites by liquid chromatography-tandem mass spectrometry (LC-MS/MS) by optimization of both the pY-immunoprecipitation (pY-IP) protocol and the LC-MS/MS parameters. Our highly sensitive method reproducibly identified more than 1000 pY sites from 8 mg of protein lysate without the need for tyrosine phosphatase inhibitor treatment. Furthermore, >30% of the identified pY sites were not assigned in the PhosphositePlus database. We further applied our method to the comparison of pY status between PC3 cells with and without treatment using the epidermal growth factor receptor (EGFR) inhibitor Erlotinib. Under Erlotinib treatment, we observed not only a decrease in well-known modes of EGFR downstream signaling but also modulations of kinases that are not relevant to the EGFR cascade, such as PTK6 and MAPK13. Our newly developed method for pY proteomics has the potential to reveal unknown pY signaling modes and to identify novel kinase anticancer targets.Deep Phosphotyrosine Proteomics by Optimization of Phosphotyrosine Enrichment and MS/MS Parameters.Abe Yuichi Y, Nagano Maiko M, Tada Asa A, Adachi Jun J, Tomonaga Takeshi Tdihydrogen phosphate (ester), Peptidomics., Tyrosine-O-phosphate, Phosphotyrosine, L-tyrosine, Tyrosine O phosphate, MOD_RES Phosphotyrosineliquid chromatography tandem mass spectroscopy, DER/faint little ball, Post Translational Amino Acid Modification, Co-Immunoprecipitations, para Tyrosine, Urogastrone Receptor, posttranslational modification, Polycomb 3 homolog, Phosphatase, Elp-B1, der, Receptors, Elp-1, Tarceva, Errb1, CP-358774, 358, protein, Protein Processing, phosphorylation, Epidermal Growth Factor Receptor Family Protein, Co Immunoprecipitation, PIG61, RC1, Phosphatases, ErbB-1, Hydrolase, Techniques, Method, epidermal growth factor-activated receptor activity, AI552599, Att-1, TGF alpha, Kinase, protein aggregate, d-egf-r, SAPK4, Post-Translational Protein, treatment, posttranslational amino acid modification, me75, LCMSMS, vanadyl hydroperoxide, Post-Translational, ErbB 1, An, 2-amino-3-(4-hydroxyphenyl)propanoic acid, 3-(p-Hydroxyphenyl)alanine, Co-Immunoprecipitation, Precipitation, K6A, K6D, Y, K6C, Epidermal Growth Factor Receptor, Posttranslational Protein Processing, Phosphomonoesterases, D17Mit170, Epidermal Growth Factor Receptor Kinase, T1, flb, allergic reaction, NEC1, Proto oncogene c ErbB 1 Protein, 11C-erlotinib, Immune, CP-358, Methodological Studies, Nec-1, TGF-alpha, Tyr, CP, disease management, Therapies, ErbB Receptor, 9030024J15Rik, D-EGFR, Precipitations, ATP Phosphotransferases, phosphatase, NISBD2, ATP, single organism signaling, DER1, LC-MS2, Therapy, Pc3, Post-Translational Modification, Elp, EGF Receptors, Wa5, l(2)09261, PC-3 cell, DEgfr, Modifications, HER Family Receptors, 2-Amino-3-(p-hydroxyphenyl)propionic acid, LC-MS/MS, N-(3-ethynylphenyl)-6, MAPK-13, para-Tyrosine, Procedure, Tl3, Tl2, AA959598, CG10079, Tyrosine, PC3, torpedo/egfr, OSI-774, Post Translational Modifications, wa2, Tyrosine O phosphate, CTRN2, Sik, erlotinibum, Urogastrone, APRO1, an-1, Erlotinib HCl, Rectachrome 1, mPc3, egfr, Transphosphorylases, HD-33, El, PTM, DER/EGFR, EGFR, EGfr, EgfR, Processing, erbB 1 Proto Oncogene Protein, dEgfr, HCl, L-isomer, 7-bis-(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-phenyl)-amine, TGF-alpha Receptor, DER flb, erbB-1 Proto-Oncogene Protein, Methodological, Torpedo/Egfr, study protocol, Methodological Study, Treatments, tks, ErbB-1 Receptor, L isomer, Degfr, Receptor Tyrosine protein Kinase erbB 1, Phosphohydrolase, Transforming-Growth Factor alpha, erlotinib, Agl, Amino Acid Modification, antagonists and inhibitors, post-translational modification, liquid chromatography-tandem mass spectroscopy, DEGFR, Phosphoric, l(2)57DEFa, l(2)05351, dihydrogen phosphate (ester), Tyrosine-O-phosphate, dEGFR, Posttranslational Modifications, L-Tyrosine, HER, DmHD-33, L-tyrosine, Hydrolases, c-ErbB-1 Protein, Posttranslational Modification, PC-3, Post-Translational Protein Modifications, D-Egf, Tksk, Procedures, ARP1B, Family Receptors, Peptidomics, C-erb, Transforming Growth Factor alpha, mor1, Transforming Growth Factor alpha Receptor, Gene, Monoester Hydrolase, mENA, protein-containing complex, EK2-6, KRT6D, KRT6C, c-erbB-1 Protein, LC-MS-MS, c erbB 1 Protein, Posttranslational, method, hPc3, CP 358, CK6D, CK6C, sensitive, CK6A, PRKM13, MOD_RES Phosphotyrosine, method used in an experiment, LC-MSMS, c-erbB, EGFr, Egfr, Post Translational Modification, Gene Products, Immune Precipitations, Studies, torpedo/Egfr, EGF receptor activity, 7-bis(2-methoxy-ethoxy)quinazoline-4-yl]-(3-ethynylphenyl)amine, Post-Translational Modifications, TOP, Low, Egf-r, sensitivity, Technique, [6, Phosphotransferase, Immune Precipitation, erbB-1, Post Translational, Modification, study, EGF-R, 11C erlotinib, inhibiteur, top, 774, Torpedo/DER, posttranslational protein modification, Transphosphorylase, OSI 774, CP358, Phosphoric Monoester Hydrolase, top/flb, Protein Modifications, Family Receptor, Post-Translational Amino Acid Modification, Study, Epidermal Growth Factor-Urogastrone, lysate, inhibidor, L Tyrosine, Posttranslational Amino Acid Modification, l(2)57EFa, Posttranslational Protein, Epidermal Growth Factor Urogastrone, 358774, l(2)57Ea, Tyrosin, Receptor Tyrosine-protein Kinase erbB-1, PC3 cell, single organism signaling., Proto-oncogene, Elp-B1RB1, inhibitors, EGF Receptor, Phosphotyrosine, cou, Epidermal Growth Factor Receptor Protein Tyrosine Kinase, Epidermal Growth Factor Receptor Family Proteins, CP 358774, protein complex, ErbB, TGF-alpha receptor activity, ERBB, dEGFR1, Proteins, Phosphorylations, Proto-Oncogene Protein, Post-Translational Protein Modification, inhibitor, function, Cell, Phosphotransferases, p38delta, Phosphomonoesterase, LC/MS/MS, Lr, native protein, epidermal growth factor receptor activity, Serk4, DmelCG10079, tirosina, Protein, Epidermal Growth Factor Receptor Protein-Tyrosine Kinase, Hydrochloride, Proto-oncogene c-ErbB-1 Protein, Erlotinib, HER Family Receptor, DER/top, Protein Modification, 7-bis(2-methoxyethoxy)quinazolin-4-amine, Data Base, antagonists, Phosphoric Monoester, CP358774, top/DER, Kinases, Post Translational Protein Processing, OSI774, phosphoric monoester hydrolase activity, ERBB1, wa-2, post-translational amino acid modification, Protein Gene Products, Phosphohydrolases, plan specification, Gene Proteins, Post Translational Protein Modification, EGF, Egf, EFG-R, MAPK 13, signalling process, Therapeutic, Errp, Tis21, Erbb, Post-Translational Protein Processing, Epidermal Growth Factor, Bra, Der, DER, HER Family, liquid chromatography tandem mass spectrometry, Treatment, Receptor, transforming growth factor-alpha receptor activity, BRK, DER/torpedo, HER1, TIS21dihydrogen phosphate (ester), Tyrosine-O-phosphate, MS2., Phosphotyrosine, L-tyrosine, Peptidomics, Tyrosine O phosphate, MS/MS, tandem MS, MOD_RES PhosphotyrosineIntervention Strategies, Co-Immunoprecipitations, Phosphotyrosine, Procedures, determination, CP 358774, N-(3-ethynylphenyl)-6, Tarceva, CP-358774, 358, Erlotinib HCl., Procedure, Cultural Backgrounds, Cell, Intervention or Procedure, Co Immunoprecipitation, Techniques, Background, OSI-774, Cultural Background, CP 358, Cultures, Method, Tyrosine O phosphate, MOD_RES Phosphotyrosine, chemical analysis, Cultural, Studies, Immune Precipitations, Hydrochloride, 7-bis(2-methoxy-ethoxy)quinazoline-4-yl]-(3-ethynylphenyl)amine, Erlotinib, erlotinibum, 7-bis(2-methoxyethoxy)quinazolin-4-amine, Technique, [6, Immune Precipitation, Backgrounds, Intervention, ethnicity, CP358774, 11C erlotinib, Cultural Beliefs, interventionDescription, OSI774, Co-Immunoprecipitation, HCl, Precipitation, 7-bis-(2-methoxy-ethoxy)-quinazolin-4-yl]-(3-ethynyl-phenyl)-amine, Interventional, 774, Cultural Relativisms, Methodological, Methodological Study, OSI 774, CP358, SURGICAL AND MEDICAL PROCEDURES, Study, 11C-erlotinib, Immune, erlotinib, CP-358, Methodological Studies, CP, dihydrogen phosphate (ester), Customs, Tyrosine-O-phosphate, Relativisms, 358774, assay, culture, Precipitations, Relativism, L-tyrosine, Cultural RelativismfalseOptimization of Phosphotyrosine ProteomicsWe optimized a procedure of phosphotyrosine enrichment with immunoprecipitation. Then the optimized procedure was applied into a global analysis of phosphotyrosine status in culture cells treated with Erlotinib. Fri Sep 29 00:00:00 BST 2017PXD005061960628152594