JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day2_Rep2.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day1_Rep3.rawhttps://storage.jpostdb.org/JPST001675/files/DDA-MS_NSC_day7.rawhttps://storage.jpostdb.org/JPST001675/files/DDA-MS_NSC_day1.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day2_Rep3.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day0_Rep4.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day3_Rep2.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day1_Rep1.rawhttps://storage.jpostdb.org/JPST001675/files/DDA-MS_NSC_day5.rawhttps://storage.jpostdb.org/JPST001675/files/DDA-MS_NSC_day3.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day0_Rep2.rawhttps://storage.jpostdb.org/JPST001675/files/DDA-MS_NSC_day0.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day1_Rep4.rawhttps://storage.jpostdb.org/JPST001675/files/DDA-MS_NSC_day2.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day1_Rep2.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day2_Rep1.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day3_Rep1.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day0_Rep1.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day2_Rep4.rawhttps://storage.jpostdb.org/JPST001675/files/DIA-MS_NSC1_day0_Rep3.rawprimaryOK200ProteomicsNana KawasakiHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST001675Yokohama city universityjPOSTHuman-induced pluripotent stem cell (iPSC)-derived neural stem cells (NSCs) have several potential applications in regenerative medicine. A deep understanding of stem cell characteristics is critical for developing appropriate products for use in the clinic. This study aimed to develop approaches for characterizing iPSC-derived NSCs. Data-independent acquisition mass spectrometry (DIA-MS) was used to obtain temporal proteomic profiles of differentiating cells. Principal component analysis of the proteome profiles allowed for the discrimination of cells cultured for different periods. Cells were characterized by Gene Ontology analysis to annotate the upregulated proteins based on their functions. We found that trophoblast glycoprotein (TPBG), a membrane glycoprotein that inhibits the Wnt/β-catenin pathway, was elevated in NSC and that silencing TPBG promoted proliferation rather than neuronal differentiation. Treatment with Wnt/β-catenin pathway activators and inhibitors showed that modulating the Wnt/β-catenin pathway is crucial for differentiation into NSC. These results suggest that the level of TPBG is critical for differentiation into NSC, and TPBG is a potentially critical quality attribute of differentiating cells. In summary, DIA-MS-based proteomics is a promising multi-attribute method for characterizing stem cell-derived products.Quantitative Proteomics for the Development and Manufacturing of Human-Induced Pluripotent Stem Cell-Derived Neural Stem Cells Using Data-Independent Acquisition Mass Spectrometry.Urasawa Takaya T, Koizumi Takumi T, Kimura Kazumasa K, Ohta Yuki Y, Kawasaki Nana NMass Spectrum Analysis, Fibroblast-Derived IPS Cells, Mass Spectrum, human being, Man (Taxonomy), Fibroblast-Derived IPS Cell, Analyses, Peptidomics, Induced Pluripotent Stem Cell, IPS, Modern, Fibroblast-Derived IPS, Stem Cells, number, Spectrometry, Fibroblast Derived IPS Cells, hiPSC, Spectrum Analyses, Fibroblast-Derived, presence, IPS Cells, Spectrum Analysis, human, Cell, Human Induced Pluripotent Stem Cells, Human, Spectroscopy, count in organism, MS, Homo sapiens, Fibroblast Derived Induced Pluripotent Stem Cells, Stem Cell, Modern Man, Neural Stem, Cells, Fibroblast-Derived Induced Pluripotent Stem 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Studies, Prot/GlycoRes+, Dwnt5, Dwnt4, 38E.16, disease management, Medicine, Br, Therapies, dWnt2, Medicines, Glycosylated Protein, wnt, Sp, Neoglycoproteins, close to, a glycoprotein, Therapy, Modern, Mother., Surface Glycoprotein, Procedure, Spectrum Analysis, results, Spectroscopy, ms(2)04138, Desmoplastic infantile astrocytoma, Colony-Forming Unit, clone 2.4, Stem Cell, Glycoproteins, D-wnt-2, Ontology Projects, DWint-1, CG4889, Mother Cell, DWnt3/5, fg, Gene Ontologies, Ontology, Ontologies, Projects, Syncytiotrophoblast, anon-WO03040301.228, Cytotrophoblasts, Stem, POF2, Glycosylated, anon-EST:Liang-2.4, F27C12_24, Fibroblast-Derived IPS, WG, F27C12.24, Spectrometry, Colony Forming Units, Methodological, Fibroblast-Derived, Features, Methodological Study, Treatments, human, animal stem cell, CG1916, Principal Component, antagonists and inhibitors, Cells, Fibroblast-Derived Induced Pluripotent Stem Cells, DIANA, Regenerative Medicines, Wg, Neural Stem Cell, Proteomes, human being, C-Glycosylated, Fibroblast-Derived IPS Cell, M6P1, Procedures, Trophoblast, Peptidomics, Induced Pluripotent Stem Cell, Cell Surface Glycoproteins, Colony-Forming Units, Progenitor Cell, Gene, Spectrum Analyses, IPS Cells, Gene Ontology Project, Gene Ontology, DWnt-3/5, NSC, Human, Regenerative, method, Homo sapiens, 5T4AG, Neural Stem, DmelCG1768, Catenin Protein, method used in an experiment, Mass, HILDA, Gene Products, Studies, Man, Progenitor, Technique, Mass Spectroscopy, Neural, study, trophoderm, WAIF1, neural stem cell, trophoblast layer, DmelCG6407, Cell Surface Glycoprotein, glycoproteines, inhibiteur, Mother, 5T4, dWnt, Wnt/Wg, O-Glycosylated Proteins, Wnt1, Human Induced Pluripotent Stem Cells, Wnt3, wnt4, wnt3, Study, wnt2, wnt1, Wnt3/5, Membrane Glycoprotein, DmelCG4698, inhibidor, Catenin Proteins, wnt5, Characteristics, glycosylated protein, DIA2, high elevation, incidence, Glykoproteine, trophoblastus, inhibitors, Colony Forming Unit, glycated protein, neuronal stem cell, l(2)rO727, Progenitor Cells, Desmoplastic astrocytoma of infancy, Proteins, Syncytiotrophoblasts, total expressed protein, Cell Surface, inhibitor, CG1768, DRF2, Cell, wgl, near to, MS, spd, iPSC, Characteristic, Fibroblast Derived Induced Pluripotent Stem Cells, massa cellularis externa, MLPLI, chemical analysis, Protein, Glycosylated Proteins, Dias, IPS Cell, Mass Spectrum Analyses, antagonists, Ontology Project, Mass Spectrum, N-Glycosylated Proteins, Protein Cd86, CG4698, stem cell, Cytotrophoblast, Dwnt-2, Dwnt-3, Understanding, Membrane, Dm DWnt2, Dm DWnt4, Protein Gene Products, plan specification, Gene Proteins, Therapeutic, approaches, Dwnt-5, Modern Man, vicinity of, Treatment, assay, DWnt-3, Human Induced Pluripotent Stem Cell, DWnt-2, DWnt-1, Gla, CG6407human being, single-organism developmental process, Fibroblast-Derived IPS Cell, Peptidomics, Induced Pluripotent Stem Cell, IPS, postnatal development, Modern, Stem Cells, number, Fibroblast Derived IPS Cells, hiPSC, growth and development, Spectrum Analyses, presence, IPS Cells, Spectrum Analysis, Cell, Human, Spectroscopy, development, count in organism, MS, iPSC, Homo sapiens, Fibroblast Derived Induced Pluripotent Stem Cells, Stem Cell, Neural Stem, Mass, Analysis, iPS cell, Man, IPS Cell, Mass Spectrum Analyses, Mass Spectroscopy, Neural, Mass Spectrum Analysis, Fibroblast-Derived IPS Cells, Mass Spectrum, Man (Taxonomy), Analyses, Fibroblast-Derived IPS, postnatal growth, Spectrometry, Fibroblast-Derived, human, DIA, Human Induced Pluripotent Stem Cells, Modern Man, Cells, Fibroblast-Derived Induced Pluripotent Stem Cells, Mass., Human Induced Pluripotent Stem Cell, growth, Neural Stem Cellsodium salt, Hac-1/Dark, ammonium formate, 13C-labeled, cadmium salt, AU023367, Meth, Phosphatase, IPS, DmelCG6829, instrument configuration, classic hairy cell leukaemia, zinc formate, Fibroblast Derived IPS Cells, hiPSC, lead salt, CREB3L1, Hydrogenchlorid, protein, magnesium formate, Cahp, Apaf-1, Deoxycholic Acid, zinc salt, 1B1, 12beta-Isomer, Phosphatases, anon-EST:Posey9, Hydrolase, ammonium tetraformate, hydrogen chloride, ethylenecarboxamide, BANF, cobalt(II) formate dihydrate, 3, Min, Apaf1, Analysis, protein aggregate, ARK, DmelCG42273, Mass Spectrum Analysis, Disodium Salt, Fibroblast-Derived IPS Cells, 3-hydroxy-5-estrane-17-carbonitrile, C79325, Man (Taxonomy), hac-1, Analyses, 2610036I19Rik, 2610510L13Rik, hypoplasia, µg, min, arc, HardwareType, mAPC, achromobacter beta-lytic protease (blp), Processed cyclic AMP-responsive element-binding protein 3-like protein 1, copper, Phosphomonoesterases, ark, T1, AI047805, fSAP152, Wasserstoffchlorid, lithium salt, Ice, ACN, sample, Acn, 3beta-Isomer, dApaf1, l(2)01103, phosphatase, ammonium (2:1) salt, Magnesium (2:1) Salt, HtsF, nickel salt, Add, ADD, D-Apaf-1, Chp, cobalt (+2) salt, Modern, anon-53Fa, Dmel_CG7826, ug, l(2)SH0173, classic hairy cell leukemia, Dmel_CG9325, Spectrum Analysis, add, Spectroscopy, 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Ovhts, aluminum salt, cesium salt, Lagodeoxycholic Acid, Gene, HtsRC, CG9325, Spectrum Analyses, ACINUS, Monoester Hydrolase, vac, protein-containing complex, Chalk, IPS Cells, Xt, 17alpha)-isomer, CG7826, NSC, Human, Buffer, formic acid, Dark/Hac-1/dApaf1, Hac1, potassium salt, Dark/Hac-1/dApaf-1, Homo sapiens, reduced, 12-dihydroxy-, Add-hts, 14C-labeled, Oasis, Tina, CG7835, Gene Products, Mass, Sodium Deoxycholate, CG42273, l(2)k14523, l(2)00634, Ovhts-RC, tiny, 12alpha)-, Man, cloruro de hidrogeno, Mass Spectroscopy, neural stem cell, hardware., 12beta Isomer, HTS-R1, Glass, Acinus, Hydrogen chloride, thallium (+1) salt, Monosodium Salt, dapaf-1, beta-Trypsin, rubidium salt, 3beta Isomer, inhibiteur, HTS-RC, dapaf, Desoxycholic Acid, dark, calcium formate, methanoic acid, Human Induced Pluripotent Stem Cells, Solution, Phosphoric Monoester Hydrolase, acinusL, inhibidor, DARK, acinusS, Viscosities, Kybella, Choleic Acid, cromium (+3) salt, dArk, Dapaf-1, CG34197, sodium formate, small, inhibitors, DmelCG43443, Monoammonium Salt, dark/dapaf-1/hac-1, Hts-RC, DYRK1, neuronal stem cell, AA960066, nickel formate, 1500003O03Rik, protein complex, 5alpha-Isomer Deoxycholic Acid, dApaf-1, Proteins, strontium formate, inhibitor, Deoxycholate, apaf1, chloridohydrogen, potassium formate, Mischung, copper (+2) salt, strontium salt, buffer, Cell, mKIAA0670, [HCl], Phosphomonoesterase, APAF1, MS, native protein, GLI3-190, Fibroblast Derived Induced Pluripotent Stem Cells, Hac-1, cupric formate, Protein, 3beta-Isomer Deoxycholic Acid, Hydrochloride, Dmel_CG34197, 5alpha, Sodium, Dyrk1, 2-Propenamide, Dark, (3alpha, aluminum formate, IPS Cell, Dark/Apaf-I, Bph, Mass Spectrum Analyses, HLB, antagonists, Achromobacter protease I, PSEC0238, AU020952, Mass Spectrum, Phosphoric Monoester, lithium formate, PRSS, underdeveloped, adducin, CC1, Tripcellim, OASIS, Dark/Dapaf-1/HAC1, Monopotassium Salt, phosphoric monoester hydrolase activity, dApaf-1/DARK/HAC-1, chromic formate, sample population, ME-IV, Phosphohydrolases, Protein Gene Products, achromopeptidase, cAMP-responsive element-binding protein 3-like protein 1, Gene Proteins, Trypure, Modern Man, calcium salt, Attention Deficit Hyperactivity Disorder, Human Induced Pluripotent Stem Cell, EST D, SCH, hydrochloric acid, dAPAF-1, 3-OHECNfalseQuantitative proteomics for efficient differentiation of human induced pluripotent stem cells into neural stem cells using data independent acquisition mass spectrometry.Human induced pluripotent stem cells were differentiated into NSC for 7 days. Cells on days 0, 1, 2, 3, 5 and 7 of differentiation (2.0 × 10⁶) were washed twice with DPBS (−) and then mixed with 100 μL of dissolution buffer consisting of 12 mM sodium deoxycholate, 12 mM N-lauroylsarcosine, 100 mM Tris-HCl pH 9.0, 1% O-GlcNAcase inhibitor, 1% Halt™ protease, and phosphatase inhibitor cocktail. The harvested cells were incubated at 95°C for 5 min and then sonicated to remove the viscosity. One hundred micrograms of protein was diluted to 2.5 μg/μL with dissolution buffer, and the proteins were reduced by adding 10 mM 2-mercaptoetanmol and incubating the mixture at 37℃ for 30 min, followed by another incubation with 20 mM acrylamide at 37℃ in the dark for 60 min. One microgram of mass spectrometry grade lysyl endopeptidase was added to the solution and incubated at 37℃ for 60 min. Then, 2 μg of trypsin was added to the solution and incubated at 37℃ for 6 h. Add an equal amount of ethyl acetate and 0.1% formic acid. After stirring, the supernatant was removed after centrifugation at 14,000 xg. The supernatant was desalted with an Oasis PriME HLB 1 cc Extraction Cartridge according to the manufacturer’s instructions. The protein fraction was dried using a Speed Vac concentrator, and the dried sample was reconstituted with 0.1% FA/3% ACN and analyzed by an Q-Exactive mass spectrometer coupled with a nanoLC instrument.Mon Jun 27 00:00:00 BST 2022PXD034925960637097202