JPOST Repositoryapplication/xmlhttps://storage.jpostdb.org/JPST001284/files/190415HN_5_ZrO2_30mMacetate_pH2.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_9_CeO2_30mMacetate_pH4.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_2_ZrO2_30mMacetate_pH5.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_14_TiO2_30mMacetate_pH5.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/181217HN_28_CeO2_acetate_pH3.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_15_TiO2_30mMacetate_pH4.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_17_TiO2_30mMacetate_pH2.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_20_GaO2_30mMacetate_pH5.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_3_ZrO2_30mMacetate_pH4.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_22_GaO2_30mMacetate_pH3.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_8_CeO2_30mMacetate_pH5.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_16_TiO2_30mMacetate_pH3.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/181217HN_26_CeO2_acetate_pH5.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/181217HN_29_CeO2_acetate_pH2.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_11_CeO2_30mMacetate_pH2.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_10_CeO2_30mMacetate_pH3.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/181217HN_27_CeO2_acetate_pH4.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_23_GaO2_30mMacetate_pH2.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_4_ZrO2_30mMacetate_pH3.wiff.scanhttps://storage.jpostdb.org/JPST001284/files/190415HN_21_GaO2_30mMacetate_pH4.wiff.scanprimaryOK200ProteomicsYasushi IshihamaHomo Sapiens (human)https://repository.jpostdb.org/entry/JPST001284Kyoto universityjPOSTWe have developed a one-step method to isolate protein C-terminal peptides from V8 protease-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, affording a digested peptide with two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the nonterminal (i.e., internal) peptide is retained, whereas the protein C-terminal peptide flows through the MOLEX column. After the optimization of the MOLEX chromatographic conditions, 1619 protein C-termini were identified from 30 μg of peptides (10 μg each, in triplicate) derived from human HeLa cells by means of nanoLC/MS/MS. When the MOLEX-isolated sample from 200 μg of HeLa peptides was further divided into six fractions by high-pH reversed-phase liquid chromatography (LC) prior to nanoLC/MS/MS, 2203 protein C-termini were identified with less than 3% contamination with internal peptides. We believe that this is the largest coverage with the highest purity reported to date in human protein C-terminomics. This fast, simple, sensitive, and selective method to isolate protein C-terminal peptides should be useful for profiling protein C-termini on a proteome-wide scale.One-Step Isolation of Protein C-Terminal Peptides from V8 Protease-Digested Proteins by Metal Oxide-Based Ligand-Exchange Chromatography.Nishida Hiroshi H, Ishihama Yasushi YLigand, GRP1/cytohesin 1, Peptide P-C, Proactivator polypeptide, Parotid double-band protein, Proteins, Pa, Saposin-A, Gene, stepk, Parotid isoelectric focusing variant protein, PROC1, PRP-3|PRP-4, Co-beta-glucosidase, Peptide, CG11633, cytohesin/GRP1, protein C (inactivator of coagulation factors Va and VIIIa), Polypeptides, Db-s, peptido, isolation, Chromatographies., GRP1, Grp1, Parotid acidic protein, Protein, 3.1.3.48, Gene Products, Sphingolipid activator protein 2, l(2)SH0323, A1 activator, PROC_HUMAN, CYH1, Salivary acidic proline-rich phosphoprotein 1|2, PRP-1|PRP-2, Pr1|Pr2, Salivary acidic proline-rich phosphoprotein 3|4, Sphingolipid activator protein 1, PRH2, peptides, Step, CG11628, Parotid proline-rich protein 1|2, Saposin-B-Val, PRH1, ligand, PTPSTEP, Dispersin, l(2)SH2 0323, CSAct, Striatum-enriched protein-tyrosine phosphatase, purification, SAP-1, SAP-2, protein C, DmelCG11628, Protein Gene Products, isolation and purification, Gene Proteins, Cerebroside sulfate activator, Component C, Neural-specific protein-tyrosine phosphatase, Protein C, Saposin-D, STEP, Saposin-C, Protein A, Saposin-B, l(2)k08110, PIF-F, Sulfatide|GM1 activator, PROC, PC, Peptid, peptidos, Polypeptide, Db-F, PIF-S, Glucosylceramidase activator, GPHPP, European Vipers, DmGluClalpha, scale tissue, DL-Aminosuccinic acid, ARMD9, SMC4, BRI2 ICD, Parotid double-band protein, C3a, Pa, Glu, GLU, Reversed-Phase Chromatography, C3b, l(3)61Ea, Azemiopinae, Dmel_CG1086, P150, Mature BRI2, clefted, glc, element, Complement C3b alpha' chain, peptide, Polypeptides, Glt1, Techniques, DmGluT1, 2-Aminoglutaric acid, peptido, Method, GRP1, Grp1, p150, glu, Sphingolipid activator protein 2, dicarboxylates, A1 activator, Complement C3d fragment, t1, dGluCl-alpha, Salivary acidic proline-rich phosphoprotein 1|2, Pr1|Pr2, D, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1, E, PPG, 1810073H04Rik, Man (Taxonomy), PRH2, peptides, BcDNA:HL07853, PRH1, plant peltate hair, PTPSTEP, Immature BRI2, DmGLUT1, As, SAP-1, SAP-2, Complement C3dg fragment, DrosGluCl, Fast, ASP, Asp, -C(O)OH, a, allergic reaction, Dmglut1, ACF, DmSMC4, AHUS5, Acf, Reverse Phase, Methodological Studies, column, Complement C3f fragment, -COOH, l(2)k08110, (+-)-Aspartic acid, Secretogranin V, sample, Chromatography, CG7535, Glutaminic acid, RCB0007, GLUCL, BRI2 intracellular domain, Complement C3c alpha' chain fragment 1, Complement C3c alpha' chain fragment 2, SIMPLE, HAT, FAST, hAPG5, GPH, Dm Asp, degree of contamination, DrosGlu-Cl-alpha, APG5-LIKE, AI255234, GluCl, Ligand, Protein E25B, subdivided, wide/broad, Metal, C3bc, Reverse Phase Chromatography, CG13908, Modern, dicarboxylate, Saposin-A, Parotid isoelectric focusing variant protein, FASTK, peltate hair., Procedure, A-lt-y-gt-, ABri23, 0074/12, APOBEC1CF, protein C (inactivator of coagulation factors Va and VIIIa), European, PIG7, 3.1.3.48, p150/Glued, anon-96Aa, atomo, atome, DYNA_DROME, CG11397, forked, DmelCG7535, Pancreatic hormone, Reversed-Phase, ASIP, DL-Asparagic acid, divided, Sphingolipid activator protein 1, DmelCG43946, Dm-GluCl, Step, CG11628, Parotid proline-rich protein 1|2, C3a anaphylatoxin, MS/MS, atoms, Striatum-enriched protein-tyrosine phosphatase, Methodological, Agouti switch protein, septate, Methodological Study, human, C3-beta-c, 2-aminobutanedioic acid, BcDNA.LD20207, carboxy, GLP-1, wide, HEL-S-62p, Reversed Phase Chromatography, Saposin-D, STEP, Saposin-C, Saposin-B, European Adders, Cells, anon-WO0118547.279, Complement C3g fragment, Polypeptide, anon-WO0118547.156, Db-F, Bri2-23, Proteomes, P150[Glued], DL-Glutamic acid, glutamic acid, dicarboxylic acid dianion, AGTI, Secretory granule endocrine protein I, human being, GRP1/cytohesin 1, Procedures, p150/glued, l(3)S007412, STK10, Gene, CG9206, PROC1, APG5L, Reverse-Phase Liquid Chromatography, Complement C3 beta chain, broad, Complement C3 alpha chain, Human, CG11633, cytohesin/GRP1, APG5, method, Db-s, AGTIL, dSMC4, Calmbp1, Homo sapiens, (R, a dicarboxylate, Pituitary polypeptide, sensitive, Adder, method used in an experiment, DmSMC4/gluon, Parotid acidic protein, Studies, Gene Products, imBRI2, DmGlu, Technique, Man, sensitivity, PROC_HUMAN, Transmembrane protein BRI, Mat, Glutamic acid, Acylation stimulating protein, BRI2, DmelCG11397, Saposin-B-Val, MS2, ligand, Dispersin, European Adder, HeLa, Agouti coat color protein, CARBOXY GROUP, l(2)SH2 0323, C-terminal peptide, mBRI2, protein C, CPAMD1, C3adesArg, Study, DrosGluCl-alpha, Component C, Neural-specific protein-tyrosine phosphatase, DmelCG6875, ATP:Fas-activated serine/threonine protein phosphotransferase activity, Reversed-Phase Liquid, PIF-F, Bri23 peptide, CT23049, PROC, peptidos, Pancreatic polypeptide, elements, Chromatographies, dicarboxylic acid dianions, PIF-S, atom, TP53I7, DrosGluCl-alpha1, BcDNA:LD20207, Viper, Peptide P-C, Proactivator polypeptide, Proteins, ACF65, ACF64, membrane form, total expressed protein, stepk, CG1086, PRP-3|PRP-4, soluble form, Co-beta-glucosidase, Peptide, Cell, Dmel_CG13908, carboxyl group, polypeptide, p150[Glued], DmelCG9206, HeLa Cell, Glutamate, Protein, European Viper, p150[glued], Reversed-Phase Liquid Chromatography, glut1, atomus, scales, GluCla, Glutaminsaeure, l(2)SH0323, Reverse-Phase Chromatography, MCPH5, tandem MS, N-terminal peptide, CYH1, BRI2C, PRP-1|PRP-2, DL-Glutaminic acid, 2-aminopentanedioic acid, Salivary acidic proline-rich phosphoprotein 3|4, P23 peptide, scale, Asps, FCP-D, CG6875, CSAct, Plp, l(2)k08819, sample population, DmelCG11628, plan specification, Protein Gene Products, Gene Proteins, Cerebroside sulfate activator, CG43946, S)-Aspartic acid, Protein C, HSE-MSF, Protein A, Modern Man, Sulfatide|GM1 activator, PC, Peptid, l(3)L1H, Gluon, -CO2H, PH, Bri, Secretogranin-5, Glucosylceramidase activatorLigand, GRP1/cytohesin 1, Peptide P-C, Proactivator polypeptide, Parotid double-band protein, Proteins, Pa, Saposin-A, Gene, stepk, Parotid isoelectric focusing variant protein, PROC1, PRP-3|PRP-4, Co-beta-glucosidase, Peptide, CG11633, cytohesin/GRP1, protein C (inactivator of coagulation factors Va and VIIIa), Polypeptides, Db-s, peptido, isolation, Chromatographies., GRP1, Grp1, Parotid acidic protein, Protein, 3.1.3.48, Gene Products, Sphingolipid activator protein 2, l(2)SH0323, A1 activator, PROC_HUMAN, CYH1, Salivary acidic proline-rich phosphoprotein 1|2, PRP-1|PRP-2, Pr1|Pr2, Salivary acidic proline-rich phosphoprotein 3|4, Sphingolipid activator protein 1, PRH2, peptides, Step, CG11628, Parotid proline-rich protein 1|2, Saposin-B-Val, PRH1, ligand, PTPSTEP, Dispersin, l(2)SH2 0323, CSAct, Striatum-enriched protein-tyrosine phosphatase, purification, SAP-1, SAP-2, protein C, DmelCG11628, Protein Gene Products, isolation and purification, Gene Proteins, Cerebroside sulfate activator, Component C, Neural-specific protein-tyrosine phosphatase, Protein C, Saposin-D, STEP, Saposin-C, Protein A, Saposin-B, l(2)k08110, PIF-F, Sulfatide|GM1 activator, PROC, PC, Peptid, peptidos, Polypeptide, Db-F, PIF-S, Glucosylceramidase activator, GPHPP, European Vipers, DmGluClalpha, DL-Aminosuccinic acid, ARMD9, SMC4, BRI2 ICD, Parotid double-band protein, C3a, Pa, Glu, GLU, C3b, l(3)61Ea, Azemiopinae, Dmel_CG1086, P150, Mature BRI2, glc, element, Complement C3b alpha' chain, peptide, Polypeptides, Glt1, Techniques, DmGluT1, 2-Aminoglutaric acid, peptido, Method, GRP1, Grp1, p150, glu, Min, Sphingolipid activator protein 2, A1 activator, Complement C3d fragment, t1, DmelCG42273, dGluCl-alpha, Salivary acidic proline-rich phosphoprotein 1|2, Pr1|Pr2, D, C3 and PZP-like alpha-2-macroglobulin domain-containing protein 1, E, PPG, 1810073H04Rik, Man (Taxonomy), PRH2, peptides, BcDNA:HL07853, PRH1, PTPSTEP, Immature BRI2, DmGLUT1, As, min, mAPC, SAP-1, purification, SAP-2, Complement C3dg fragment, DrosGluCl, ASP, Asp, -C(O)OH, AI047805, a, isolation and purification, allergic reaction, Dmglut1, ACF, DmSMC4, AHUS5, Acf, Methodological Studies, column, Complement C3f fragment, -COOH, l(2)k08110, (+-)-Aspartic acid, Secretogranin V, sample, CG7535, Glutaminic acid, GLUCL, BRI2 intracellular domain, Complement C3c alpha' chain fragment 1, Complement C3c alpha' chain fragment 2, SIMPLE, HAT, hAPG5, GPH, Dm Asp, DrosGlu-Cl-alpha, APG5-LIKE, AI255234, GluCl, Ligand, Protein E25B, Data Set, Metal, C3bc, CG13908, Modern, Dmel_CG7826, Saposin-A, Parotid isoelectric focusing variant protein, Procedure, A-lt-y-gt-, ABri23, results, 0074/12, APOBEC1CF, protein C (inactivator of coagulation factors Va and VIIIa), Dm1, European, PIG7, 3.1.3.48, p150/Glued, anon-96Aa, atomo, atome, DYNA_DROME, Dmel_CG7835, CG11397, DmelCG7535, Mnb, MNB, Pancreatic hormone, ASIP, DL-Asparagic acid, Sphingolipid activator protein 1, DmelCG43946, Dm-GluCl, Step, CG11628, Parotid proline-rich protein 1|2, C3a anaphylatoxin, atoms, Striatum-enriched protein-tyrosine phosphatase, Methodological, Agouti switch protein, AW124434, Methodological Study, human, C3-beta-c, 2-aminobutanedioic acid, BcDNA.LD20207, carboxy, GLP-1, HEL-S-62p, Saposin-D, STEP, Saposin-C, Saposin-B, European Adders, Cells, anon-WO0118547.279, Complement C3g fragment, Polypeptide, anon-WO0118547.156, Db-F, Bri2-23, P150[Glued], DL-Glutamic acid, glutamic acid, AGTI, Secretory granule endocrine protein I, human being, GRP1/cytohesin 1, Procedures, p150/glued, l(3)S007412, Gene, CG9206, PROC1, APG5L, Complement C3 beta chain, Complement C3 alpha chain, CG7826, Human, CG11633, cytohesin/GRP1, APG5, method, Db-s, AGTIL, dSMC4, Calmbp1, Homo sapiens, (R, isolation, Pituitary polypeptide, sensitive, Adder, method used in an experiment, DmSMC4/gluon, Parotid acidic protein, Studies, Gene Products, CG7835, imBRI2, CG42273, DmGlu, Technique, Man, sensitivity, PROC_HUMAN, Transmembrane protein BRI, Mat, Glutamic acid, Acylation stimulating protein, BRI2, DmelCG11397, Saposin-B-Val, ligand, Dispersin, European Adder, HeLa, Agouti coat color protein, CARBOXY GROUP, l(2)SH2 0323, C-terminal peptide, mBRI2, protein C, CPAMD1, C3adesArg, Study, DrosGluCl-alpha, Component C, Neural-specific protein-tyrosine phosphatase, DmelCG6875, PIF-F, Bri23 peptide, CT23049, PROC, peptidos, Pancreatic polypeptide, elements, Chromatographies, PIF-S, atom, TP53I7, DrosGluCl-alpha1, DYRK1, BcDNA:LD20207, Viper, Peptide P-C, Proactivator polypeptide, Proteins, ACF65, ACF64, membrane form, stepk, CG1086, PRP-3|PRP-4, soluble form, Co-beta-glucosidase, Peptide, Cell, Dmel_CG13908, carboxyl group, polypeptide, p150[Glued], DmelCG9206, HeLa Cell, Glutamate, Protein, European Viper, p150[glued], glut1, atomus, Dyrk1, GluCla, Glutaminsaeure, l(2)SH0323, MCPH5, N-terminal peptide, CYH1, AU020952, BRI2C, PRP-1|PRP-2, DL-Glutaminic acid, 2-aminopentanedioic acid, Salivary acidic proline-rich phosphoprotein 3|4, P23 peptide, Asps, FCP-D, CC1, CG6875, CSAct, Plp, l(2)k08819, sample population, DmelCG11628, ME-IV, plan specification, Protein Gene Products, Gene Proteins, Cerebroside sulfate activator, N-terminal peptide., CG43946, S)-Aspartic acid, Protein C, HSE-MSF, Protein A, Modern Man, Sulfatide|GM1 activator, PC, Peptid, l(3)L1H, Gluon, -CO2H, PH, Bri, Secretogranin-5, Glucosylceramidase activatorfalseOne-step Isolation of Protein C-terminal Peptides from Protease-digested Proteins by Metal Oxide-based Ligand Exchange ChromatographyWe have developed a one-step method to isolate protein C-terminal peptides from V8-digested proteins by metal oxide-based ligand-exchange (MOLEX) chromatography. V8 protease cleaves the C-terminal side of Asp and Glu, and the resulting digested peptide has two carboxy groups at the C-terminus, whereas the protein C-terminal peptide has only one α-carboxy group. In MOLEX chromatography, a stable chelate is formed between dicarboxylates and metal atoms, so that the non-terminal (i.e., internal) peptide is retained, but the protein C-terminal peptide flows through the MOLEX column. By optimizing the conditions of V8 protease digestion and MOLEX chromatographic isolation, a total of 1619 protein C-termini from 30 μg of peptides derived from human HeLa cells could be identified in 10 min of the isolation step. Furthermore, using 200 µg of peptide, we identified 2202 protein C-termini, which was the largest data set with the least amount of sample in protein C-terminomics. These results indicated that this method is unprecedentedly simple and sensitive for protein C-terminal peptide enrichment. Tue Dec 28 00:00:00 GMT 2021PXD027960960634962382