JP4558297B2 - Detection / separation / identification method of expressed trace protein / peptide - Google Patents
Detection / separation / identification method of expressed trace protein / peptide Download PDFInfo
- Publication number
- JP4558297B2 JP4558297B2 JP2003342681A JP2003342681A JP4558297B2 JP 4558297 B2 JP4558297 B2 JP 4558297B2 JP 2003342681 A JP2003342681 A JP 2003342681A JP 2003342681 A JP2003342681 A JP 2003342681A JP 4558297 B2 JP4558297 B2 JP 4558297B2
- Authority
- JP
- Japan
- Prior art keywords
- protein
- peptide
- fluorescent
- hplc
- fluorescence
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Expired - Lifetime
Links
- 108090000623 proteins and genes Proteins 0.000 title claims description 134
- 102000004169 proteins and genes Human genes 0.000 title claims description 129
- 108090000765 processed proteins & peptides Proteins 0.000 title claims description 110
- 238000000034 method Methods 0.000 title claims description 65
- 238000001514 detection method Methods 0.000 title claims description 30
- 238000000926 separation method Methods 0.000 title claims description 28
- 238000004128 high performance liquid chromatography Methods 0.000 claims description 49
- 239000003153 chemical reaction reagent Substances 0.000 claims description 47
- 102000004196 processed proteins & peptides Human genes 0.000 claims description 30
- 239000000523 sample Substances 0.000 claims description 23
- 238000004458 analytical method Methods 0.000 claims description 17
- 238000001917 fluorescence detection Methods 0.000 claims description 16
- 102000004190 Enzymes Human genes 0.000 claims description 15
- 108090000790 Enzymes Proteins 0.000 claims description 15
- 229940088598 enzyme Drugs 0.000 claims description 15
- 239000012634 fragment Substances 0.000 claims description 14
- 238000006243 chemical reaction Methods 0.000 claims description 13
- 238000012360 testing method Methods 0.000 claims description 13
- 102000007079 Peptide Fragments Human genes 0.000 claims description 11
- 108010033276 Peptide Fragments Proteins 0.000 claims description 11
- 108090000631 Trypsin Proteins 0.000 claims description 10
- 102000004142 Trypsin Human genes 0.000 claims description 10
- 238000004949 mass spectrometry Methods 0.000 claims description 9
- 239000004094 surface-active agent Substances 0.000 claims description 9
- 239000012588 trypsin Substances 0.000 claims description 9
- 150000001413 amino acids Chemical class 0.000 claims description 8
- 238000012916 structural analysis Methods 0.000 claims description 8
- 230000007071 enzymatic hydrolysis Effects 0.000 claims description 7
- 238000006047 enzymatic hydrolysis reaction Methods 0.000 claims description 7
- 238000012795 verification Methods 0.000 claims description 6
- 125000000524 functional group Chemical group 0.000 claims description 5
- 230000014509 gene expression Effects 0.000 claims description 5
- 230000007062 hydrolysis Effects 0.000 claims description 5
- 238000006460 hydrolysis reaction Methods 0.000 claims description 5
- 108091005804 Peptidases Proteins 0.000 claims description 4
- 102000035195 Peptidases Human genes 0.000 claims description 4
- 239000003398 denaturant Substances 0.000 claims description 4
- 238000005342 ion exchange Methods 0.000 claims description 4
- 238000003011 reverse-phase partition HPLC Methods 0.000 claims description 4
- 238000004007 reversed phase HPLC Methods 0.000 claims description 4
- 108090000317 Chymotrypsin Proteins 0.000 claims description 3
- 239000007864 aqueous solution Substances 0.000 claims description 3
- 239000012472 biological sample Substances 0.000 claims description 3
- 238000005194 fractionation Methods 0.000 claims description 3
- 238000002523 gelfiltration Methods 0.000 claims description 3
- 238000012544 monitoring process Methods 0.000 claims description 3
- 235000019833 protease Nutrition 0.000 claims description 3
- 238000001962 electrophoresis Methods 0.000 claims description 2
- 235000018102 proteins Nutrition 0.000 description 78
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 21
- JXLHNMVSKXFWAO-UHFFFAOYSA-N azane;7-fluoro-2,1,3-benzoxadiazole-4-sulfonic acid Chemical compound N.OS(=O)(=O)C1=CC=C(F)C2=NON=C12 JXLHNMVSKXFWAO-UHFFFAOYSA-N 0.000 description 21
- 239000000243 solution Substances 0.000 description 21
- XUJNEKJLAYXESH-REOHCLBHSA-N L-Cysteine Chemical compound SC[C@H](N)C(O)=O XUJNEKJLAYXESH-REOHCLBHSA-N 0.000 description 15
- RJEYFWVHUXSWTA-UHFFFAOYSA-N 4-chloro-n-[2-(dimethylamino)ethyl]-2,1,3-benzoxadiazole-7-sulfonamide Chemical compound CN(C)CCNS(=O)(=O)C1=CC=C(Cl)C2=NON=C12 RJEYFWVHUXSWTA-UHFFFAOYSA-N 0.000 description 14
- DTQVDTLACAAQTR-UHFFFAOYSA-N Trifluoroacetic acid Chemical compound OC(=O)C(F)(F)F DTQVDTLACAAQTR-UHFFFAOYSA-N 0.000 description 14
- 239000000872 buffer Substances 0.000 description 14
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 14
- 235000018417 cysteine Nutrition 0.000 description 14
- 238000001212 derivatisation Methods 0.000 description 14
- 238000004366 reverse phase liquid chromatography Methods 0.000 description 12
- 230000035945 sensitivity Effects 0.000 description 12
- 150000001875 compounds Chemical class 0.000 description 11
- 239000012071 phase Substances 0.000 description 11
- 230000035484 reaction time Effects 0.000 description 11
- FAPWRFPIFSIZLT-UHFFFAOYSA-M Sodium chloride Chemical compound [Na+].[Cl-] FAPWRFPIFSIZLT-UHFFFAOYSA-M 0.000 description 10
- BTBUEUYNUDRHOZ-UHFFFAOYSA-N Borate Chemical compound [O-]B([O-])[O-] BTBUEUYNUDRHOZ-UHFFFAOYSA-N 0.000 description 9
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 9
- -1 N, N-Dimethylamino Chemical group 0.000 description 9
- 239000000203 mixture Substances 0.000 description 9
- 238000010828 elution Methods 0.000 description 8
- BDAGIHXWWSANSR-UHFFFAOYSA-N methanoic acid Natural products OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 8
- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 description 7
- 230000001965 increasing effect Effects 0.000 description 7
- UMCMPZBLKLEWAF-BCTGSCMUSA-N 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate Chemical compound C([C@H]1C[C@H]2O)[C@H](O)CC[C@]1(C)[C@@H]1[C@@H]2[C@@H]2CC[C@H]([C@@H](CCC(=O)NCCC[N+](C)(C)CCCS([O-])(=O)=O)C)[C@@]2(C)[C@@H](O)C1 UMCMPZBLKLEWAF-BCTGSCMUSA-N 0.000 description 6
- YMWUJEATGCHHMB-UHFFFAOYSA-N Dichloromethane Chemical compound ClCCl YMWUJEATGCHHMB-UHFFFAOYSA-N 0.000 description 6
- ZMANZCXQSJIPKH-UHFFFAOYSA-N Triethylamine Chemical compound CCN(CC)CC ZMANZCXQSJIPKH-UHFFFAOYSA-N 0.000 description 6
- 238000012790 confirmation Methods 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 6
- 238000002330 electrospray ionisation mass spectrometry Methods 0.000 description 6
- 239000003480 eluent Substances 0.000 description 6
- 230000005284 excitation Effects 0.000 description 6
- 238000004255 ion exchange chromatography Methods 0.000 description 6
- 238000005259 measurement Methods 0.000 description 6
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 6
- 241000700159 Rattus Species 0.000 description 5
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 5
- PZBFGYYEXUXCOF-UHFFFAOYSA-N TCEP Chemical compound OC(=O)CCP(CCC(O)=O)CCC(O)=O PZBFGYYEXUXCOF-UHFFFAOYSA-N 0.000 description 5
- 235000001014 amino acid Nutrition 0.000 description 5
- 238000013418 fluorescence derivatisation Methods 0.000 description 5
- 102000034287 fluorescent proteins Human genes 0.000 description 5
- 108091006047 fluorescent proteins Proteins 0.000 description 5
- 238000004895 liquid chromatography mass spectrometry Methods 0.000 description 5
- 239000011148 porous material Substances 0.000 description 5
- 239000011541 reaction mixture Substances 0.000 description 5
- 239000011780 sodium chloride Substances 0.000 description 5
- 125000003396 thiol group Chemical group [H]S* 0.000 description 5
- 150000003573 thiols Chemical class 0.000 description 5
- OSWFIVFLDKOXQC-UHFFFAOYSA-N 4-(3-methoxyphenyl)aniline Chemical compound COC1=CC=CC(C=2C=CC(N)=CC=2)=C1 OSWFIVFLDKOXQC-UHFFFAOYSA-N 0.000 description 4
- OKKJLVBELUTLKV-MZCSYVLQSA-N Deuterated methanol Chemical compound [2H]OC([2H])([2H])[2H] OKKJLVBELUTLKV-MZCSYVLQSA-N 0.000 description 4
- 238000005481 NMR spectroscopy Methods 0.000 description 4
- 108010026552 Proteome Proteins 0.000 description 4
- 230000015572 biosynthetic process Effects 0.000 description 4
- 239000003638 chemical reducing agent Substances 0.000 description 4
- 238000004587 chromatography analysis Methods 0.000 description 4
- 150000001944 cysteine derivatives Chemical class 0.000 description 4
- 235000019253 formic acid Nutrition 0.000 description 4
- 229960000789 guanidine hydrochloride Drugs 0.000 description 4
- PJJJBBJSCAKJQF-UHFFFAOYSA-N guanidinium chloride Chemical compound [Cl-].NC(N)=[NH2+] PJJJBBJSCAKJQF-UHFFFAOYSA-N 0.000 description 4
- NOESYZHRGYRDHS-UHFFFAOYSA-N insulin Chemical compound N1C(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(NC(=O)CN)C(C)CC)CSSCC(C(NC(CO)C(=O)NC(CC(C)C)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CCC(N)=O)C(=O)NC(CC(C)C)C(=O)NC(CCC(O)=O)C(=O)NC(CC(N)=O)C(=O)NC(CC=2C=CC(O)=CC=2)C(=O)NC(CSSCC(NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2C=CC(O)=CC=2)NC(=O)C(CC(C)C)NC(=O)C(C)NC(=O)C(CCC(O)=O)NC(=O)C(C(C)C)NC(=O)C(CC(C)C)NC(=O)C(CC=2NC=NC=2)NC(=O)C(CO)NC(=O)CNC2=O)C(=O)NCC(=O)NC(CCC(O)=O)C(=O)NC(CCCNC(N)=N)C(=O)NCC(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC=CC=3)C(=O)NC(CC=3C=CC(O)=CC=3)C(=O)NC(C(C)O)C(=O)N3C(CCC3)C(=O)NC(CCCCN)C(=O)NC(C)C(O)=O)C(=O)NC(CC(N)=O)C(O)=O)=O)NC(=O)C(C(C)CC)NC(=O)C(CO)NC(=O)C(C(C)O)NC(=O)C1CSSCC2NC(=O)C(CC(C)C)NC(=O)C(NC(=O)C(CCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(NC(=O)C(N)CC=1C=CC=CC=1)C(C)C)CC1=CN=CN1 NOESYZHRGYRDHS-UHFFFAOYSA-N 0.000 description 4
- 230000009257 reactivity Effects 0.000 description 4
- 238000003786 synthesis reaction Methods 0.000 description 4
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 3
- OJSCBKGRGMBEEW-UHFFFAOYSA-N 3-fluorobenzene-1,2-diamine Chemical compound NC1=CC=CC(F)=C1N OJSCBKGRGMBEEW-UHFFFAOYSA-N 0.000 description 3
- YXFVVABEGXRONW-UHFFFAOYSA-N Toluene Chemical compound CC1=CC=CC=C1 YXFVVABEGXRONW-UHFFFAOYSA-N 0.000 description 3
- UREBDLICKHMUKA-CXSFZGCWSA-N dexamethasone Chemical compound C1CC2=CC(=O)C=C[C@]2(C)[C@]2(F)[C@@H]1[C@@H]1C[C@@H](C)[C@@](C(=O)CO)(O)[C@@]1(C)C[C@@H]2O UREBDLICKHMUKA-CXSFZGCWSA-N 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- RWSXRVCMGQZWBV-WDSKDSINSA-N glutathione Chemical compound OC(=O)[C@@H](N)CCC(=O)N[C@@H](CS)C(=O)NCC(O)=O RWSXRVCMGQZWBV-WDSKDSINSA-N 0.000 description 3
- 238000002347 injection Methods 0.000 description 3
- 239000007924 injection Substances 0.000 description 3
- 150000002500 ions Chemical class 0.000 description 3
- 238000002372 labelling Methods 0.000 description 3
- 238000004012 multidimensional HPLC Methods 0.000 description 3
- 229960001723 oxytocin Drugs 0.000 description 3
- 239000000843 powder Substances 0.000 description 3
- 239000000741 silica gel Substances 0.000 description 3
- 229910002027 silica gel Inorganic materials 0.000 description 3
- 229960000553 somatostatin Drugs 0.000 description 3
- 239000000126 substance Substances 0.000 description 3
- 229910052717 sulfur Inorganic materials 0.000 description 3
- 229960003726 vasopressin Drugs 0.000 description 3
- YBJHBAHKTGYVGT-ZKWXMUAHSA-N (+)-Biotin Chemical compound N1C(=O)N[C@@H]2[C@H](CCCCC(=O)O)SC[C@@H]21 YBJHBAHKTGYVGT-ZKWXMUAHSA-N 0.000 description 2
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- PGZIDERTDJHJFY-UHFFFAOYSA-N 4-fluoro-7-nitro-2,1,3-benzoxadiazole Chemical compound [O-][N+](=O)C1=CC=C(F)C2=NON=C12 PGZIDERTDJHJFY-UHFFFAOYSA-N 0.000 description 2
- XROXHZMRDABMHS-UHFFFAOYSA-N 7-fluoro-2,1,3-benzoxadiazole-4-sulfonamide Chemical compound NS(=O)(=O)C1=CC=C(F)C2=NON=C12 XROXHZMRDABMHS-UHFFFAOYSA-N 0.000 description 2
- HFDKKNHCYWNNNQ-YOGANYHLSA-N 75976-10-2 Chemical compound C([C@@H](C(=O)N[C@@H]([C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(N)=O)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CC(O)=O)NC(=O)[C@H](C)NC(=O)[C@H](C)NC(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CCSC)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H]1N(CCC1)C(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(O)=O)NC(=O)CNC(=O)[C@H]1N(CCC1)C(=O)[C@H](CC=1C=CC(O)=CC=1)NC(=O)[C@@H](NC(=O)[C@H]1N(CCC1)C(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CC(C)C)NC(=O)[C@H]1N(CCC1)C(=O)[C@H](C)N)C(C)C)[C@@H](C)O)C1=CC=C(O)C=C1 HFDKKNHCYWNNNQ-YOGANYHLSA-N 0.000 description 2
- QGZKDVFQNNGYKY-UHFFFAOYSA-N Ammonia Chemical compound N QGZKDVFQNNGYKY-UHFFFAOYSA-N 0.000 description 2
- USFZMSVCRYTOJT-UHFFFAOYSA-N Ammonium acetate Chemical compound N.CC(O)=O USFZMSVCRYTOJT-UHFFFAOYSA-N 0.000 description 2
- 239000005695 Ammonium acetate Substances 0.000 description 2
- ATRRKUHOCOJYRX-UHFFFAOYSA-N Ammonium bicarbonate Chemical compound [NH4+].OC([O-])=O ATRRKUHOCOJYRX-UHFFFAOYSA-N 0.000 description 2
- 229910000013 Ammonium bicarbonate Inorganic materials 0.000 description 2
- UXVMQQNJUSDDNG-UHFFFAOYSA-L Calcium chloride Chemical compound [Cl-].[Cl-].[Ca+2] UXVMQQNJUSDDNG-UHFFFAOYSA-L 0.000 description 2
- VEXZGXHMUGYJMC-UHFFFAOYSA-M Chloride anion Chemical compound [Cl-] VEXZGXHMUGYJMC-UHFFFAOYSA-M 0.000 description 2
- HEDRZPFGACZZDS-UHFFFAOYSA-N Chloroform Chemical compound ClC(Cl)Cl HEDRZPFGACZZDS-UHFFFAOYSA-N 0.000 description 2
- ZRALSGWEFCBTJO-UHFFFAOYSA-N Guanidine Chemical compound NC(N)=N ZRALSGWEFCBTJO-UHFFFAOYSA-N 0.000 description 2
- 102000004877 Insulin Human genes 0.000 description 2
- 108090001061 Insulin Proteins 0.000 description 2
- FFFHZYDWPBMWHY-VKHMYHEASA-N L-homocysteine Chemical compound OC(=O)[C@@H](N)CCS FFFHZYDWPBMWHY-VKHMYHEASA-N 0.000 description 2
- 241000244206 Nematoda Species 0.000 description 2
- 102400000050 Oxytocin Human genes 0.000 description 2
- 101800000989 Oxytocin Proteins 0.000 description 2
- XNOPRXBHLZRZKH-UHFFFAOYSA-N Oxytocin Natural products N1C(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CC(C)C)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C(C(C)CC)NC(=O)C1CC1=CC=C(O)C=C1 XNOPRXBHLZRZKH-UHFFFAOYSA-N 0.000 description 2
- 102000018886 Pancreatic Polypeptide Human genes 0.000 description 2
- 108010076181 Proinsulin Proteins 0.000 description 2
- 108010000605 Ribosomal Proteins Proteins 0.000 description 2
- 102000002278 Ribosomal Proteins Human genes 0.000 description 2
- 102000005157 Somatostatin Human genes 0.000 description 2
- 108010056088 Somatostatin Proteins 0.000 description 2
- 101000983124 Sus scrofa Pancreatic prohormone precursor Proteins 0.000 description 2
- 102100036407 Thioredoxin Human genes 0.000 description 2
- GXBMIBRIOWHPDT-UHFFFAOYSA-N Vasopressin Natural products N1C(=O)C(CC=2C=C(O)C=CC=2)NC(=O)C(N)CSSCC(C(=O)N2C(CCC2)C(=O)NC(CCCN=C(N)N)C(=O)NCC(N)=O)NC(=O)C(CC(N)=O)NC(=O)C(CCC(N)=O)NC(=O)C1CC1=CC=CC=C1 GXBMIBRIOWHPDT-UHFFFAOYSA-N 0.000 description 2
- 108010004977 Vasopressins Proteins 0.000 description 2
- 102000002852 Vasopressins Human genes 0.000 description 2
- 239000002253 acid Substances 0.000 description 2
- 230000002378 acidificating effect Effects 0.000 description 2
- 229940043376 ammonium acetate Drugs 0.000 description 2
- 235000019257 ammonium acetate Nutrition 0.000 description 2
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 2
- 239000001099 ammonium carbonate Substances 0.000 description 2
- VZTDIZULWFCMLS-UHFFFAOYSA-N ammonium formate Chemical compound [NH4+].[O-]C=O VZTDIZULWFCMLS-UHFFFAOYSA-N 0.000 description 2
- KBZOIRJILGZLEJ-LGYYRGKSSA-N argipressin Chemical compound C([C@H]1C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@@H](C(N[C@@H](CC=2C=CC(O)=CC=2)C(=O)N1)=O)N)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCN=C(N)N)C(=O)NCC(N)=O)C1=CC=CC=C1 KBZOIRJILGZLEJ-LGYYRGKSSA-N 0.000 description 2
- 239000001110 calcium chloride Substances 0.000 description 2
- 229910001628 calcium chloride Inorganic materials 0.000 description 2
- 125000003178 carboxy group Chemical group [H]OC(*)=O 0.000 description 2
- 125000000151 cysteine group Chemical group N[C@@H](CS)C(=O)* 0.000 description 2
- 230000007423 decrease Effects 0.000 description 2
- 230000003247 decreasing effect Effects 0.000 description 2
- 229960003957 dexamethasone Drugs 0.000 description 2
- 238000010586 diagram Methods 0.000 description 2
- 230000000694 effects Effects 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 238000002189 fluorescence spectrum Methods 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 108010017007 glucose-regulated proteins Proteins 0.000 description 2
- 229910052736 halogen Inorganic materials 0.000 description 2
- 150000002367 halogens Chemical group 0.000 description 2
- 229940125396 insulin Drugs 0.000 description 2
- 210000004153 islets of langerhan Anatomy 0.000 description 2
- 230000014759 maintenance of location Effects 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000011259 mixed solution Substances 0.000 description 2
- 230000007935 neutral effect Effects 0.000 description 2
- XNOPRXBHLZRZKH-DSZYJQQASA-N oxytocin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CSSC[C@H](N)C(=O)N1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CC(C)C)C(=O)NCC(N)=O)=O)[C@@H](C)CC)C1=CC=C(O)C=C1 XNOPRXBHLZRZKH-DSZYJQQASA-N 0.000 description 2
- 210000000496 pancreas Anatomy 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000002360 preparation method Methods 0.000 description 2
- 150000003839 salts Chemical class 0.000 description 2
- 229910052711 selenium Inorganic materials 0.000 description 2
- 239000011669 selenium Substances 0.000 description 2
- NHXLMOGPVYXJNR-ATOGVRKGSA-N somatostatin Chemical compound C([C@H]1C(=O)N[C@H](C(N[C@@H](CO)C(=O)N[C@@H](CSSC[C@@H](C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C=CC=CC=2)C(=O)N[C@@H](CC=2C3=CC=CC=C3NC=2)C(=O)N[C@@H](CCCCN)C(=O)N[C@H](C(=O)N1)[C@@H](C)O)NC(=O)CNC(=O)[C@H](C)N)C(O)=O)=O)[C@H](O)C)C1=CC=CC=C1 NHXLMOGPVYXJNR-ATOGVRKGSA-N 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 238000003756 stirring Methods 0.000 description 2
- 239000006228 supernatant Substances 0.000 description 2
- 108060008226 thioredoxin Proteins 0.000 description 2
- 229940094937 thioredoxin Drugs 0.000 description 2
- 210000001519 tissue Anatomy 0.000 description 2
- RIOQSEWOXXDEQQ-UHFFFAOYSA-N triphenylphosphine Chemical compound C1=CC=CC=C1P(C=1C=CC=CC=1)C1=CC=CC=C1 RIOQSEWOXXDEQQ-UHFFFAOYSA-N 0.000 description 2
- 208000001072 type 2 diabetes mellitus Diseases 0.000 description 2
- AGQDOUOEJSDDQH-BJQYWQRQSA-N (2S)-N-[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-1-[[(2S)-4-amino-1-[[(2S)-4-amino-1-[[(2S)-1-[[2-[(2S)-2-[[(2S)-1-[[(2S)-1-[(2S)-2-[(2S)-2-[[(2S,3R)-1-[[(2S)-4-amino-1-[[(2S)-1-[[2-[[(2S)-1-[[(2S)-4-amino-1-[[(2S,3R)-1-[[(2S)-1-amino-3-(4-hydroxyphenyl)-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-2-oxoethyl]amino]-3-methyl-1-oxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-hydroxy-1-oxobutan-2-yl]carbamoyl]pyrrolidine-1-carbonyl]pyrrolidin-1-yl]-4-methyl-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]carbamoyl]pyrrolidin-1-yl]-2-oxoethyl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-3-hydroxy-1-oxopropan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]amino]-3-methyl-1-oxobutan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-1-oxo-3-phenylpropan-2-yl]amino]-1,4-dioxobutan-2-yl]amino]-1-oxopropan-2-yl]amino]-4-methyl-1-oxopentan-2-yl]amino]-5-carbamimidamido-1-oxopentan-2-yl]-2-[[(2S,3R)-2-[[(2S)-2-[[(4R,7S,10S,13S,16S,19R)-16-(2-amino-2-oxoethyl)-19-[[(2S)-2,6-diaminohexanoyl]amino]-7,13-bis[(1R)-1-hydroxyethyl]-10-methyl-6,9,12,15,18-pentaoxo-1,2-dithia-5,8,11,14,17-pentazacycloicosane-4-carbonyl]amino]propanoyl]amino]-3-hydroxybutanoyl]amino]pentanediamide Chemical compound CC(C)C[C@H](NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](CO)NC(=O)[C@H](CO)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@@H](NC(=O)[C@H](CC(C)C)NC(=O)[C@H](Cc1ccccc1)NC(=O)[C@H](CC(N)=O)NC(=O)[C@H](C)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCCNC(N)=N)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](C)NC(=O)[C@@H]1CSSC[C@H](NC(=O)[C@@H](N)CCCCN)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](C)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1)[C@@H](C)O)C(C)C)C(=O)NCC(=O)N1CCC[C@H]1C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC(C)C)C(=O)N1CCC[C@H]1C(=O)N1CCC[C@H]1C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](C(C)C)C(=O)NCC(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](Cc1ccc(O)cc1)C(N)=O AGQDOUOEJSDDQH-BJQYWQRQSA-N 0.000 description 1
- FIOJWGRGPONADF-UHFFFAOYSA-N (sulfinylamino)benzene Chemical compound O=S=NC1=CC=CC=C1 FIOJWGRGPONADF-UHFFFAOYSA-N 0.000 description 1
- TVNJKAZMPQNGGE-UHFFFAOYSA-N 1,2,3-benzoselenadiazole Chemical compound C1=CC=C2[se]N=NC2=C1 TVNJKAZMPQNGGE-UHFFFAOYSA-N 0.000 description 1
- FNQJDLTXOVEEFB-UHFFFAOYSA-N 1,2,3-benzothiadiazole Chemical group C1=CC=C2SN=NC2=C1 FNQJDLTXOVEEFB-UHFFFAOYSA-N 0.000 description 1
- MVLWYDGJBGPXOL-UHFFFAOYSA-N 139332-66-4 Chemical compound C12=NON=C2C([N+](=O)[O-])=CC=C1N1CCNCC1 MVLWYDGJBGPXOL-UHFFFAOYSA-N 0.000 description 1
- OHRVDMZCBKNZTG-UHFFFAOYSA-N 2,2,2-trichloro-n-[(7-fluoro-2,1,3-benzoxadiazol-4-yl)sulfonyl]acetamide Chemical compound FC1=CC=C(S(=O)(=O)NC(=O)C(Cl)(Cl)Cl)C2=NON=C12 OHRVDMZCBKNZTG-UHFFFAOYSA-N 0.000 description 1
- VHBHPSHPLSAXTE-UHFFFAOYSA-M 2-[(4-chloro-2,1,3-benzoselenadiazol-7-yl)sulfonylamino]ethyl-trimethylazanium;chloride Chemical compound [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(Cl)C2=N[se]N=C12 VHBHPSHPLSAXTE-UHFFFAOYSA-M 0.000 description 1
- BLKGIXBLRWZLKS-UHFFFAOYSA-N 2-[methyl-(4-nitro-2,1,3-benzoxadiazol-7-yl)amino]acetyl chloride Chemical compound ClC(=O)CN(C)C1=CC=C([N+]([O-])=O)C2=NON=C12 BLKGIXBLRWZLKS-UHFFFAOYSA-N 0.000 description 1
- VSZWLDAGOXQHNB-UHFFFAOYSA-M 2-aminoethyl(trimethyl)azanium;chloride Chemical compound [Cl-].C[N+](C)(C)CCN VSZWLDAGOXQHNB-UHFFFAOYSA-M 0.000 description 1
- FKVIYERFOLSTTM-UHFFFAOYSA-N 2-fluoro-n-phenylacetamide Chemical compound FCC(=O)NC1=CC=CC=C1 FKVIYERFOLSTTM-UHFFFAOYSA-N 0.000 description 1
- YITNZTMNWIIAGA-UHFFFAOYSA-N 4-chloro-2,1,3-benzoxadiazole-7-sulfonic acid Chemical compound OS(=O)(=O)C1=CC=C(Cl)C2=NON=C12 YITNZTMNWIIAGA-UHFFFAOYSA-N 0.000 description 1
- BPPRLMZEVZSIIJ-UHFFFAOYSA-N 4-chloro-2,1,3-benzoxadiazole-7-sulfonyl chloride Chemical compound ClC1=CC=C(S(Cl)(=O)=O)C2=NON=C12 BPPRLMZEVZSIIJ-UHFFFAOYSA-N 0.000 description 1
- ACBMUXCCQGEWFT-UHFFFAOYSA-N 4-fluoro-2,1,3-benzoselenadiazole Chemical compound FC1=CC=CC2=N[se]N=C12 ACBMUXCCQGEWFT-UHFFFAOYSA-N 0.000 description 1
- WHPJUWNVXIBGMN-UHFFFAOYSA-N 4-fluoro-2,1,3-benzothiadiazole Chemical compound FC1=CC=CC2=NSN=C12 WHPJUWNVXIBGMN-UHFFFAOYSA-N 0.000 description 1
- 102100022600 40S ribosomal protein S3a Human genes 0.000 description 1
- 229920001817 Agar Polymers 0.000 description 1
- QGZKDVFQNNGYKY-UHFFFAOYSA-O Ammonium Chemical compound [NH4+] QGZKDVFQNNGYKY-UHFFFAOYSA-O 0.000 description 1
- VHUUQVKOLVNVRT-UHFFFAOYSA-N Ammonium hydroxide Chemical compound [NH4+].[OH-] VHUUQVKOLVNVRT-UHFFFAOYSA-N 0.000 description 1
- 108010020366 Arginine kinase Proteins 0.000 description 1
- 241000894006 Bacteria Species 0.000 description 1
- 238000009010 Bradford assay Methods 0.000 description 1
- 101100178679 Caenorhabditis elegans hsp-1 gene Proteins 0.000 description 1
- 102100029968 Calreticulin Human genes 0.000 description 1
- 108090000549 Calreticulin Proteins 0.000 description 1
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 description 1
- 241000588724 Escherichia coli Species 0.000 description 1
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 1
- 108010024636 Glutathione Proteins 0.000 description 1
- 101000679249 Homo sapiens 40S ribosomal protein S3a Proteins 0.000 description 1
- VEXZGXHMUGYJMC-UHFFFAOYSA-N Hydrochloric acid Chemical compound Cl VEXZGXHMUGYJMC-UHFFFAOYSA-N 0.000 description 1
- 206010022489 Insulin Resistance Diseases 0.000 description 1
- 102000036770 Islet Amyloid Polypeptide Human genes 0.000 description 1
- 108010041872 Islet Amyloid Polypeptide Proteins 0.000 description 1
- 102000013460 Malate Dehydrogenase Human genes 0.000 description 1
- 108010026217 Malate Dehydrogenase Proteins 0.000 description 1
- CHJJGSNFBQVOTG-UHFFFAOYSA-N N-methyl-guanidine Natural products CNC(N)=N CHJJGSNFBQVOTG-UHFFFAOYSA-N 0.000 description 1
- GRYLNZFGIOXLOG-UHFFFAOYSA-N Nitric acid Chemical compound O[N+]([O-])=O GRYLNZFGIOXLOG-UHFFFAOYSA-N 0.000 description 1
- 108010068204 Peptide Elongation Factors Proteins 0.000 description 1
- 102000002508 Peptide Elongation Factors Human genes 0.000 description 1
- OAICVXFJPJFONN-UHFFFAOYSA-N Phosphorus Chemical compound [P] OAICVXFJPJFONN-UHFFFAOYSA-N 0.000 description 1
- 102000004191 Ribosomal protein L1 Human genes 0.000 description 1
- 108090000792 Ribosomal protein L1 Proteins 0.000 description 1
- PMZURENOXWZQFD-UHFFFAOYSA-L Sodium Sulfate Chemical compound [Na+].[Na+].[O-]S([O-])(=O)=O PMZURENOXWZQFD-UHFFFAOYSA-L 0.000 description 1
- NINIDFKCEFEMDL-UHFFFAOYSA-N Sulfur Chemical compound [S] NINIDFKCEFEMDL-UHFFFAOYSA-N 0.000 description 1
- 108010090932 Vitellogenins Proteins 0.000 description 1
- ALGMFJBNLVFXDO-UHFFFAOYSA-M [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(Cl)C2=NSN=C12 Chemical compound [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(Cl)C2=NSN=C12 ALGMFJBNLVFXDO-UHFFFAOYSA-M 0.000 description 1
- ITEJETSYVUHXKW-UHFFFAOYSA-M [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(F)C2=NON=C12 Chemical compound [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(F)C2=NON=C12 ITEJETSYVUHXKW-UHFFFAOYSA-M 0.000 description 1
- OCYIDQIWRKKYSA-UHFFFAOYSA-M [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(F)C2=NSN=C12 Chemical compound [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(F)C2=NSN=C12 OCYIDQIWRKKYSA-UHFFFAOYSA-M 0.000 description 1
- QJBZLMUBGSNTMV-UHFFFAOYSA-M [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(F)C2=N[se]N=C12 Chemical compound [Cl-].C[N+](C)(C)CCNS(=O)(=O)C1=CC=C(F)C2=N[se]N=C12 QJBZLMUBGSNTMV-UHFFFAOYSA-M 0.000 description 1
- NEHKXDZBVVZXOE-UHFFFAOYSA-N [Se](=O)=O.C(C)O Chemical compound [Se](=O)=O.C(C)O NEHKXDZBVVZXOE-UHFFFAOYSA-N 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 239000004480 active ingredient Substances 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000008272 agar Substances 0.000 description 1
- 150000001412 amines Chemical class 0.000 description 1
- 125000000539 amino acid group Chemical group 0.000 description 1
- 125000003277 amino group Chemical group 0.000 description 1
- 229910021529 ammonia Inorganic materials 0.000 description 1
- 239000000908 ammonium hydroxide Substances 0.000 description 1
- 239000002280 amphoteric surfactant Substances 0.000 description 1
- 238000005349 anion exchange Methods 0.000 description 1
- 125000000129 anionic group Chemical group 0.000 description 1
- 239000003945 anionic surfactant Substances 0.000 description 1
- IOJUPLGTWVMSFF-UHFFFAOYSA-N benzothiazole Chemical compound C1=CC=C2SC=NC2=C1 IOJUPLGTWVMSFF-UHFFFAOYSA-N 0.000 description 1
- 229960002685 biotin Drugs 0.000 description 1
- 235000020958 biotin Nutrition 0.000 description 1
- 239000011616 biotin Substances 0.000 description 1
- 239000008280 blood Substances 0.000 description 1
- 210000004369 blood Anatomy 0.000 description 1
- 229960004015 calcitonin Drugs 0.000 description 1
- 239000002775 capsule Substances 0.000 description 1
- 229910052799 carbon Inorganic materials 0.000 description 1
- 230000015556 catabolic process Effects 0.000 description 1
- 239000003054 catalyst Substances 0.000 description 1
- 125000002091 cationic group Chemical group 0.000 description 1
- 239000003093 cationic surfactant Substances 0.000 description 1
- 238000005119 centrifugation Methods 0.000 description 1
- 239000003795 chemical substances by application Substances 0.000 description 1
- 229960002376 chymotrypsin Drugs 0.000 description 1
- 239000000470 constituent Substances 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- 238000001816 cooling Methods 0.000 description 1
- 238000006731 degradation reaction Methods 0.000 description 1
- 230000000593 degrading effect Effects 0.000 description 1
- 206010012601 diabetes mellitus Diseases 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- SWSQBOPZIKWTGO-UHFFFAOYSA-N dimethylaminoamidine Natural products CN(C)C(N)=N SWSQBOPZIKWTGO-UHFFFAOYSA-N 0.000 description 1
- AXZAYXJCENRGIM-UHFFFAOYSA-J dipotassium;tetrabromoplatinum(2-) Chemical compound [K+].[K+].[Br-].[Br-].[Br-].[Br-].[Pt+2] AXZAYXJCENRGIM-UHFFFAOYSA-J 0.000 description 1
- 238000001035 drying Methods 0.000 description 1
- 230000002255 enzymatic effect Effects 0.000 description 1
- 238000002474 experimental method Methods 0.000 description 1
- IRXSLJNXXZKURP-UHFFFAOYSA-N fluorenylmethyloxycarbonyl chloride Chemical compound C1=CC=C2C(COC(=O)Cl)C3=CC=CC=C3C2=C1 IRXSLJNXXZKURP-UHFFFAOYSA-N 0.000 description 1
- ZFKJVJIDPQDDFY-UHFFFAOYSA-N fluorescamine Chemical compound C12=CC=CC=C2C(=O)OC1(C1=O)OC=C1C1=CC=CC=C1 ZFKJVJIDPQDDFY-UHFFFAOYSA-N 0.000 description 1
- 239000008103 glucose Substances 0.000 description 1
- 230000009229 glucose formation Effects 0.000 description 1
- 229960003180 glutathione Drugs 0.000 description 1
- 229960004198 guanidine Drugs 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 230000002440 hepatic effect Effects 0.000 description 1
- 230000003301 hydrolyzing effect Effects 0.000 description 1
- 230000002209 hydrophobic effect Effects 0.000 description 1
- 125000002887 hydroxy group Chemical group [H]O* 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 230000001939 inductive effect Effects 0.000 description 1
- 238000005040 ion trap Methods 0.000 description 1
- 238000002955 isolation Methods 0.000 description 1
- 238000001819 mass spectrum Methods 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000691 measurement method Methods 0.000 description 1
- DILRJUIACXKSQE-UHFFFAOYSA-N n',n'-dimethylethane-1,2-diamine Chemical compound CN(C)CCN DILRJUIACXKSQE-UHFFFAOYSA-N 0.000 description 1
- XFQLOSBBYVMBGT-UHFFFAOYSA-N n,n-dimethyl-7-piperazin-1-yl-2,1,3-benzoxadiazole-4-sulfonamide Chemical compound C12=NON=C2C(S(=O)(=O)N(C)C)=CC=C1N1CCNCC1 XFQLOSBBYVMBGT-UHFFFAOYSA-N 0.000 description 1
- CXDCWGIDRFMMPX-UHFFFAOYSA-N n-(2-fluoro-6-nitrophenyl)acetamide Chemical compound CC(=O)NC1=C(F)C=CC=C1[N+]([O-])=O CXDCWGIDRFMMPX-UHFFFAOYSA-N 0.000 description 1
- JJXFIZSGNXTASD-UHFFFAOYSA-N n-[(7-fluoro-2,1,3-benzoxadiazol-4-yl)sulfonyl]acetamide Chemical compound CC(=O)NS(=O)(=O)C1=CC=C(F)C2=NON=C12 JJXFIZSGNXTASD-UHFFFAOYSA-N 0.000 description 1
- 229910017604 nitric acid Inorganic materials 0.000 description 1
- 239000002736 nonionic surfactant Substances 0.000 description 1
- 235000015097 nutrients Nutrition 0.000 description 1
- 239000012074 organic phase Substances 0.000 description 1
- 229910052760 oxygen Inorganic materials 0.000 description 1
- 238000012540 peak fractionation analysis Methods 0.000 description 1
- 125000001095 phosphatidyl group Chemical group 0.000 description 1
- 102000051624 phosphatidylethanolamine binding protein Human genes 0.000 description 1
- 108700021017 phosphatidylethanolamine binding protein Proteins 0.000 description 1
- 229910001487 potassium perchlorate Inorganic materials 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 238000002203 pretreatment Methods 0.000 description 1
- 239000000047 product Substances 0.000 description 1
- 238000012509 protein identification method Methods 0.000 description 1
- 229940024999 proteolytic enzymes for treatment of wounds and ulcers Drugs 0.000 description 1
- 238000010791 quenching Methods 0.000 description 1
- 230000000171 quenching effect Effects 0.000 description 1
- 239000012488 sample solution Substances 0.000 description 1
- 238000010845 search algorithm Methods 0.000 description 1
- 238000011896 sensitive detection Methods 0.000 description 1
- 230000035939 shock Effects 0.000 description 1
- 239000000377 silicon dioxide Substances 0.000 description 1
- 239000011734 sodium Substances 0.000 description 1
- 229910052938 sodium sulfate Inorganic materials 0.000 description 1
- 235000011152 sodium sulphate Nutrition 0.000 description 1
- 239000002904 solvent Substances 0.000 description 1
- BDHFUVZGWQCTTF-UHFFFAOYSA-N sulfonic acid Chemical group OS(=O)=O BDHFUVZGWQCTTF-UHFFFAOYSA-N 0.000 description 1
- 239000011593 sulfur Substances 0.000 description 1
- HIFJUMGIHIZEPX-UHFFFAOYSA-N sulfuric acid;sulfur trioxide Chemical compound O=S(=O)=O.OS(O)(=O)=O HIFJUMGIHIZEPX-UHFFFAOYSA-N 0.000 description 1
- YBBRCQOCSYXUOC-UHFFFAOYSA-N sulfuryl dichloride Chemical compound ClS(Cl)(=O)=O YBBRCQOCSYXUOC-UHFFFAOYSA-N 0.000 description 1
- 238000000539 two dimensional gel electrophoresis Methods 0.000 description 1
Images
Landscapes
- Other Investigation Or Analysis Of Materials By Electrical Means (AREA)
- Investigating Or Analyzing Non-Biological Materials By The Use Of Chemical Means (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Description
本発明は、微量の発現タンパク質及び／又はペプチドの検出・分離・同定方法に関するものであり、更に詳しくは、生体において遺伝子の発現により産生される微量の発現タンパク質及び／又はペプチドを簡便な方法で、高感度に検出し、同定することを可能とする新規な発現タンパク質及び／又はペプチドの検出・分離・同定方法、及びその同定システムに関するものである。
本発明は、ポストゲノム時代において重要な役割を果たすことが期待される、発現タンパク質及び／又はペプチドを網羅的に解析するプロテオーム技術における新しい検出・分離・同定手法を提供するものとして有用である。
The present invention relates to a method for detecting / separating / identifying a minute amount of expressed protein and / or peptide. More specifically, the present invention relates to a minute amount of expressed protein and / or peptide produced by gene expression in a living body by a simple method. The present invention relates to a novel expression protein and / or peptide detection / separation / identification method and identification system that enable detection and identification with high sensitivity.
The present invention is useful as a new detection / separation / identification technique in proteome technology that comprehensively analyzes expressed proteins and / or peptides that are expected to play an important role in the post-genomic era.
ポストゲノム時代において重要な課題は、遺伝子を介して発現する発現タンパク質／ペプチドの微量検出とその分離・同定である。従来、この課題達成のために、２次元電氣泳動後のペプチドフィンガープリント法が汎用されてきた（非特許文献１参照）。しかし、この方法は、煩雑な操作のために該方法の再現性に難点があった。この難点を克服する手法として、最近、多次元高速液体クロマトグラフィー（多次元ＨＰＬＣ）による分離・同定法、或いはＩＣＡＴによる手法が提案されている（非特許文献２参照）。 An important issue in the post-genomic era is the detection and separation / identification of expressed proteins / peptides expressed via genes. Conventionally, in order to achieve this problem, a peptide fingerprint method after two-dimensional electrophoresis has been widely used (see Non-Patent Document 1). However, this method has difficulty in reproducibility of the method due to complicated operations. As a technique for overcoming this difficulty, a separation / identification method by multidimensional high performance liquid chromatography (multidimensional HPLC) or a technique by ICAT has been recently proposed (see Non-Patent Document 2).
これらのうち、タンパク質／ペプチドを、直接、多次元ＨＰＬＣで分離・同定する方法は、全てのタンパク質／ペプチドを同時に処理するために、多大な労力と時間を要するという欠点がある。また、ＩＣＡＴによる手法は、チオール含有タンパク質／ペプチドのチオール基をｉｓｏｔｏｐｅ−ｃｏｄｅｄ ａｆｆｉｎｉｔｙ ｔａｇｓ （ＩＣＡＴ） ｒｅａｇｅｎｔで標識した後、それをビオチン結合カラムにて捕集し、これら全てを酵素水解し、得られたペプチドフラグメント混合物をＨＰＬＣで分離、質量分析計（ＭＳ）にて質量分析し、タンパク質／ペプチドを網羅的に解析しようとするものである。しかし、この方法は、チオール含有タンパク質／ペプチドの全てを酵素水解するため、大量に存在する目的以外のタンパク質／ペプチドのフラグメントが、目的とする微量タンパク質／ペプチドのフラグメントの検出及びその同定を妨害する、と言う欠点があり、当技術分野においては、更なる方法のブレークスルーが必要とされていた。 Among these, the method of directly separating and identifying proteins / peptides by multidimensional HPLC has a drawback that it takes a lot of labor and time to process all the proteins / peptides simultaneously. Moreover, the method by ICAT is obtained by labeling the thiol group of a thiol-containing protein / peptide with isotopic-coded affinity tags (ICAT) reagent, collecting it with a biotin-binding column, and hydrolyzing all of them. The peptide fragment mixture was separated by HPLC and mass analysis was performed with a mass spectrometer (MS) to comprehensively analyze proteins / peptides. However, this method enzymatically hydrolyzes all of the thiol-containing proteins / peptides, so that a large amount of non-target protein / peptide fragments interferes with the detection and identification of the desired trace protein / peptide fragments. , And there has been a need in the art for further method breakthroughs.
このような状況の中で、本発明者らは、上記従来技術に鑑みて、上記従来技術における諸問題を抜本的に解決することを目標として鋭意研究を積み重ねた結果、従来法と異なり、被験試料中の蛍光標識可能なタンパク質及び／又はペプチドのみを蛍光選択的に分離した後、これを酵素水解に付し、分画した蛍光画分を質量分析、データベース照合、構造解析に供することにより、従来法では検出不可能であった微量の発現タンパク質及び／又はペプチドを高感度に検出し、同定することができることを見出し、本発明を完成するに至った。 In such a situation, in view of the above prior art, the present inventors have conducted extensive research with the goal of drastically solving the problems in the above prior art. After only fluorescently labelable protein and / or peptide in the sample is selectively separated by fluorescence, this is subjected to enzymatic hydrolysis, and the fractionated fluorescent fraction is subjected to mass spectrometry, database verification, and structural analysis. The inventors have found that trace amounts of expressed proteins and / or peptides that could not be detected by conventional methods can be detected and identified with high sensitivity, and the present invention has been completed.
本発明は、遺伝子を介して発現する微量の発現タンパク質及び／又はペプチドを、簡便な測定手法で、高感度に検出・分離・同定することを可能とする上記発現タンパク質及び／又はペプチドの微量検出・分離・同定方法を提供することを目的とするものである。
また、本発明は、上記微量検出・分離・同定方法に使用する微量の発現タンパク質及び／又はペプチドを、高感度で検出・分離・同定するための発現タンパク質及び／又はペプチド同定システムを提供することを目的とするものである。
更に、本発明は、従来法では検出することができなかった、遺伝子を介して発現する微量の発現タンパク質及び／又はペプチドを超高感度で検出・分離・同定することを可能とする新しい分析方法及び手段を提供することを目的とする。
The present invention provides a trace amount detection of the expressed protein and / or peptide that enables highly sensitive detection, separation and identification of a small amount of expressed protein and / or peptide expressed via a gene by a simple measurement technique. -It is intended to provide a separation / identification method.
In addition, the present invention provides an expressed protein and / or peptide identification system for detecting, separating and identifying trace amounts of expressed proteins and / or peptides used in the above-described trace amount detection, separation and identification methods with high sensitivity. It is intended.
Furthermore, the present invention provides a new analytical method that enables detection, separation, and identification of a very small amount of expressed protein and / or peptide expressed through a gene that could not be detected by a conventional method with ultrahigh sensitivity. And to provide a means.
上記課題を解決するための本発明は、以下の技術的手段から構成される。
（１）被験試料中の発現微量タンパク質及び／又はペプチドを高感度に検出・分離・同定する方法であって、（ａ）被験試料中のタンパク質及び／又はペプチドを該タンパク質及び／又はペプチドとの反応で蛍光を発する親水性の蛍光試薬で標識する、（ｂ）それを１次元又は２次元のＨＰＬＣ／蛍光検出により、その蛍光分画を捕集する、（ｃ）上記蛍光分画を酵素水解に付する、（ｄ）それを第二段階のＨＰＬＣ／蛍光検出により、その蛍光クロマトグラムを得ると共に、その画分を質量分析又はＭＳ／ＭＳ分析に付し、データベース照合、構造解析に供して発現タンパク質及び／又はペプチドの同定を行うことを特徴とする上記発現タンパク質及び／又はペプチドの検出・分離・同定方法。
（２）タンパク質及び／又はペプチド試料の水溶液に、官能基特異的蛍光試薬を加え、場合により、界面活性剤及び／又はタンパク変性剤を加えあるいは加えることなく、タンパク質及び／又はペプチドを蛍光標識する、前記（１）に記載の方法。
（３）蛍光標識したタンパク質及び／又はペプチド試料を蛍光検出器付きイオン交換カラムＨＰＬＣ、逆相分配ＨＰＬＣ、ゲル濾過ＨＰＬＣ、又は電気泳動による分離手段に付し、蛍光をモニターしながらそのピーク分画を捕集する、前記（１）に記載の方法。
（４）蛍光分画を、ペプチダーゼ、トリプシン、又はキモトリプシンのタンパク質分解酵素を用いて酵素水解する、前記（１）に記載の方法。
（５）酵素水解物を蛍光検出器付き逆相ＨＰＬＣに付し、蛍光ピークを検出すると共に、蛍光標識フラグメント及び蛍光非標識フラグメントの質量分析又はＭＳ／ＭＳ分析を行う、前記（１）に記載の方法。
（６）質量分析又はＭＳ／ＭＳ分析に付して得られた各フラグメントのイオン分子量情報を、コンピューターによるタンパク質及び／又はペブチドフラグメントデータベースと照合し、構造解析して、酵素水解以前のタンパク質及び／又はペプチドの同定を行う、前記（１）に記載の方法。
（７）被験試料が、生体試料から採取したタンパク質及び／又はペプチド試料である、前記（１）に記載の方法。
（８）タンパク質及び／又はペプチドフラグメント情報、及び蛍光試薬で標識したアミノ酸の情報を含んだデータベースを用いてデータベース照合する、前記（１）に記載の方法。
（９）前記（１）から（８）のいずれかに記載の方法に使用する発現微量タンパク質及び／又はペプチド検出・分離・同定システムであって、被験試料のタンパク質及び／又はペプチドを蛍光試薬で標識するための第一反応器、蛍光試薬で標識した蛍光誘導体を蛍光分画するための１次元又は２次元の蛍光検出器付きＨＰＬＣ、蛍光分画を酵素水解するための第二反応器、酵素水解物の蛍光標識フラグメントを蛍光検出するための第二段階の蛍光検出器付きＨＰＬＣ、これに接続する質量分析装置、及び蛍光試薬で標識したアミノ酸の情報を含んだデータベースを搭載した構造解析装置を構成要素として含むことを特徴とする上記検出・分離・同定システム。
（１０）上記第一反応器、１次元又は２次元の蛍光検出器付きＨＰＬＣ、第二反応器、第二段階の蛍光検出器付きＨＰＬＣを直列に配置してなる、前記（９）に記載のシステム。
The present invention for solving the above-described problems comprises the following technical means.
(1) A method for detecting, separating, and identifying expressed protein and / or peptide in a test sample with high sensitivity, wherein (a) the protein and / or peptide in the test sample is combined with the protein and / or peptide. Label with a hydrophilic fluorescent reagent that fluoresces in the reaction, (b) collect the fluorescent fraction by one-dimensional or two-dimensional HPLC / fluorescence detection, (c) enzymatically hydrolyze the fluorescent fraction. (D) The fluorescence chromatogram is obtained by HPLC / fluorescence detection in the second stage, and the fraction is subjected to mass spectrometry or MS / MS analysis, and subjected to database verification and structural analysis. A method for detecting / separating / identifying the expressed protein and / or peptide, wherein the expressed protein and / or peptide is identified.
(2) A functional group-specific fluorescent reagent is added to an aqueous solution of a protein and / or peptide sample, and in some cases, the protein and / or peptide is fluorescently labeled with or without adding a surfactant and / or a protein denaturant The method according to (1) above.
(3) Fluorescently labeled protein and / or peptide samples are subjected to ion exchange column HPLC with fluorescence detector, reverse phase partition HPLC, gel filtration HPLC, or electrophoresis separation means, and peak fractionation is monitored while monitoring fluorescence. The method as described in said (1) which collects.
(4) The method according to (1) above, wherein the fluorescent fraction is subjected to enzymatic hydrolysis using a peptidase, trypsin, or chymotrypsin proteolytic enzyme.
(5) The enzyme hydrolyzate is subjected to reverse phase HPLC with a fluorescence detector to detect a fluorescence peak, and mass spectrometry or MS / MS analysis of a fluorescence labeled fragment and a fluorescence unlabeled fragment is performed. the method of.
(6) The ionic molecular weight information of each fragment obtained by mass spectrometry or MS / MS analysis is collated with a protein and / or peptide fragment database by a computer, the structure is analyzed, and the protein before enzyme hydrolysis The method according to (1) above, wherein peptide identification is performed.
(7) The method according to (1) above, wherein the test sample is a protein and / or peptide sample collected from a biological sample.
(8) The method according to (1) above, wherein database verification is performed using a database including protein and / or peptide fragment information and information on amino acids labeled with a fluorescent reagent.
(9) An expression trace protein and / or peptide detection / separation / identification system used in the method according to any one of (1) to (8) above, wherein a protein and / or peptide of a test sample is detected with a fluorescent reagent First reactor for labeling, HPLC with one- or two-dimensional fluorescence detector for fluorescent fractionation of fluorescent derivatives labeled with fluorescent reagents, second reactor for enzymatic hydrolysis of fluorescent fractions, enzyme the second stage of the fluorescence detector with HPLC, mass spectrometer connected thereto, and structural analysis equipment equipped with a database that contains information of the labeled amino acid with a fluorescent reagent for fluorescence detection of a fluorescent label fragment hydrolysates The above detection / separation / identification system characterized by comprising:
(10) The first reactor, the one-dimensional or two-dimensional HPLC with fluorescence detector, the second reactor, and the second stage HPLC with fluorescence detector are arranged in series, as described in (9) above system.
次に、本発明について更に詳細に説明する。
本発明は、上記従来法の難点を克服するためになされたものであり、１）微量の発現タンパク質／ペプチドを蛍光試薬で標識し、２）それをＨＰＬＣ／蛍光検出にて第一段の分離・蛍光検出を行い、３）その蛍光分画（コントロール試料と比べて、被験試料に特異的に増減する蛍光画分）のみを捕集後、酵素水解し、それを第二段のＨＰＬＣ／蛍光検出にて分離し、蛍光ピークの確認を行った後、ＨＰＬＣ／ＭＳに付し、蛍光標識タンパク質／ペプチドフラグメントの同定を行い、当該微量タンパク質／ペプチドの特定を行う方法に関するものである。尚、タンパク質／ペプチド試料が純度の高い場合には、第一段のＨＰＬＣ／蛍光検出による分離を省くことができる。本発明の方法は、従来法と異なり、蛍光標識可能なタンパク質／ペプチドのみを特異的に抽出し、検出・同定することができるという特徴を有し、微量の発現タンパク質／ペプチドを特定するために尤も相応しい方法である。
Next, the present invention will be described in more detail.
The present invention has been made to overcome the above-mentioned difficulties of the conventional methods. 1) A small amount of expressed protein / peptide is labeled with a fluorescent reagent, and 2) it is separated in the first stage by HPLC / fluorescence detection. -Fluorescence detection is performed. 3) After collecting only the fluorescence fraction (fluorescence fraction that increases or decreases specifically in the test sample compared to the control sample), the enzyme is hydrolyzed, and then the second stage HPLC / fluorescence is collected. The present invention relates to a method of separating by detection and confirming a fluorescence peak, followed by HPLC / MS, identifying a fluorescently labeled protein / peptide fragment, and specifying the trace protein / peptide. If the protein / peptide sample is high in purity, the first-stage separation by HPLC / fluorescence detection can be omitted. Unlike the conventional method, the method of the present invention is characterized in that only a fluorescently labelable protein / peptide can be specifically extracted, detected and identified, and for identifying a minute amount of expressed protein / peptide. It is a reasonable method.
本発明では、被験試料として、生体から採取したあらゆる種類のタンパク質及び／又はペプチドを含む試料が対象とされる。本発明の方法では、被験試料中の微量の発現タンパク質／ペプチドを蛍光試薬で標識し、蛍光誘導体化するが、この場合、タンパク質／ペプチド水溶液に、官能基特異的蛍光試薬を加え、場合により、界面活性剤及び／又はタンパク変性剤を加え、発現タンパク質／ペプチドを定量的に誘導体化することが重要である。即ち、本発明では、タンパク質／ペプチド試料の水溶液に、界面活性剤と場合によっては還元剤を添加し、これに官能基特異的蛍光試薬を加え、必要により、加温することにより、タンパク質及び／又はペプチドを蛍光標識する。本発明では、上記界面活性剤として、非イオン性、陰イオン性、陽イオン性及び両イオン性界面活性剤が用いられる。また、本発明では、上記還元剤として、好適には、Ｔｒｉｓ（２−ｃａｒｂｏｘｙｅｔｈｙｌ）ｐｈｏｓｐｈｉｎｅ、ｔｒｉｂｕｔｙｌｐｈｏｓｐｈｉｎｅが用いられるが、これらに制限されるものではなく、同効のものであれば同様に使用することができる。 In the present invention, a sample containing all kinds of proteins and / or peptides collected from a living body is a target sample. In the method of the present invention, a small amount of expressed protein / peptide in a test sample is labeled with a fluorescent reagent and fluorescent derivatized. In this case, a functional group-specific fluorescent reagent is added to the protein / peptide aqueous solution. It is important to add a surfactant and / or protein denaturant to quantitatively derivatize the expressed protein / peptide. That is, in the present invention, a surfactant and optionally a reducing agent are added to an aqueous protein / peptide sample solution, a functional group-specific fluorescent reagent is added thereto, and if necessary, the protein and / or the Alternatively, the peptide is fluorescently labeled. In the present invention, nonionic, anionic, cationic and amphoteric surfactants are used as the surfactant. Further, in the present invention, Tris (2-carboxyethyl) phosphine and triphenylphosphine are preferably used as the reducing agent. However, the reducing agent is not limited to these and may be used in the same manner as long as they have the same effect. Can do.
本発明において、上記官能基特異的蛍光試薬として、（例えば、４−Ｆｌｕｏｒｏ−７−ｎｉｔｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＮＢＤ−Ｆ）、 ５−（Ｎ，Ｎ−Ｄｉｍｅｔｈｙｌａｍｉｎｏ）ｎａｐｈｔｈａｌｅｎｅ−１−ｓｕｌｆｏｎｙｌ ｃｈｌｏｒｉｄｅ （ＤＮＳ−ＣＬ）、Ｏｒｔｈｏｐｈｔｈａｌｄｅｈｙｄｅ（ＯＰＡ）、Ｆｌｕｏｒｅｓｃａｍｉｎｅ、 ９−Ｆｌｕｏｒｅｎｙｌｍｅｔｈｙｌ ｃｈｌｏｒｏｆｏｒｍａｔｅ （ＦＭＯＣ）等のアミノ基特異的蛍光試薬、Ａｍｍｏｎｉｕｍ ７−ｆｌｕｏｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ−４−ｓｕｌｆｏｎａｔｅ（ＳＢＤ−Ｆ）、４−（Ａｍｉｎｏｓｕｌｆｏｎｙｌ）−７−ｆｌｕｏｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＡＢＤ−Ｆ）、４−（Ａｃｅｔｙｌａｍｉｎｏｓｕｌｆｏｎｙｌ）−７−ｆｌｕｏｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＡｃＡＢＤ−Ｆ）、４−Ｆｌｕｏｒｏ−７−ｔｒｉｃｈｌｏｒｏａｃｅｔｙｌａｍｉｎｏｓｕｌｆｏｎｙｌ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＴｃＡｃＡＢＤ−Ｆ）、ｍｏｎｏｂｒｏｍｏｂｉｍａｎｅ等のチオール基特異的蛍光試薬、４−Ｎｉｔｒｏ−７−Ｎ−ｐｉｐｅｒａｚｉｎｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＮＢＤ−ＰＺ）、４−Ｎ，Ｎ−Ｄｉｍｅｔｈｙｌａｍｉｎｏｓｕｌｆｏｎｙｌ−７−Ｎ−ｐｉｐｅｒａｚｉｎｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＤＢＤ−ＰＺ）と縮合剤との組み合わせによるカルボキシル基特異的蛍光試薬、又は、４−（Ｎ−Ｃｈｌｏｒｏｆｏｒｍｙｌｍｅｔｈｙｌ−Ｎ−ｍｅｔｈｙｌ）ａｍｉｎｏ−７−ｎｉｔｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＮＢＤ−ＣＯＣＬ）等の水酸基用蛍光試薬）が例示されるが、これらに制限されない。 In the present invention, the functional group-specific fluorescent reagent includes (for example, 4-Fluoro-7-nitro-2,1,3-benzazodiazole (NBD-F), 5- (N, N-Dimethylamino) naphthalene-1- Amino group-specific fluorescent reagents such as sulfuryl chloride (DNS-CL), Orthophthaldehyde (OPA), Fluorescamine, 9-Fluorenylmethyl chloroformate (FMOC), Ammonium 7-fluorz, 4-sulolate, F), 4- (Aminosulfonyl) -7-fluoro-2,1,3-benzoxadiazole (AB) -F), 4- (Acetylaminosulfonyl) -7-fluoro-2,1,3-benzoxadiazole (AcABD-F), 4-Fluoro-7-trichloroacetylaminosulfonyl-2,1,3-benzoxadiazole (TcFacAmBondomoBm) Thiol group specific fluorescent reagent, 4-Nitro-7-N-piperazino-2,1,3-benzazodiazole (NBD-PZ), 4-N, N-Dimethylaminosulfonyl-7-N-piperazino-2,1,3 A carboxyl group-specific fluorescent reagent by a combination of -benzoxazole (DBD-PZ) and a condensing agent, or 4- (N-Chlo) (formylmethyl-N-methyl) amino-7-nitro-2,1,3-benzazodiazole (NBD-COCL) and the like), but is not limited thereto.
本発明においては、必要により、加温する（例えば、３０−１００℃、望ましくは４０−７０℃で１０−３００分間、望ましくは６０−１８０分間）ことにより、タンパク質／ペプチドを蛍光標識する。その後、反応液のほぼ全量を蛍光検出器付きイオン交換カラムＨＰＬＣ、又は逆相分配ＨＰＬＣ、又はゲル濾過ＨＰＬＣに付し、蛍光をモニターしながらピーク分画を分取する。この場合、蛍光検出は、標識蛍光体の励起・蛍光波長に相当する波長に設定して行う。例えば、ＮＢＤ−Ｆ、又はＳＢＤ−Ｆで標識した場合には、励起波長４８０ｎｍ或いは３８０ｎｍ、励起波長５２０ｎｍ又は５０５ｎｍに設定する。イオン交換ＨＰＬＣの場合には、塩、例えば、食塩、硫酸ナトリウム、過塩素酸カリウム、酢酸アンモニウムなど、望ましくは酢酸アンモニウムのような揮発性の塩を段階的に増量し、それぞれの画分を得る。この画分そのもの又はこの画分を濃縮・乾固した試料を、酵素水解に付す。酵素としては、各種ペプチダーゼ、トリプシン、キモトリプシンなど、適宜のタンパク質分解酵素が用いられる。この際、酵素カラムを接続してオンラインで酵素水解を行うこともできる。 In the present invention, if necessary, the protein / peptide is fluorescently labeled by heating (for example, 30-100 ° C., desirably 40-70 ° C., 10-300 minutes, desirably 60-180 minutes). Thereafter, almost the entire amount of the reaction solution is subjected to ion exchange column HPLC equipped with a fluorescence detector, reverse phase partition HPLC, or gel filtration HPLC, and a peak fraction is collected while monitoring fluorescence. In this case, fluorescence detection is performed at a wavelength corresponding to the excitation / fluorescence wavelength of the labeled phosphor. For example, when labeled with NBD-F or SBD-F, the excitation wavelength is set to 480 nm or 380 nm, and the excitation wavelength is set to 520 nm or 505 nm. In the case of ion exchange HPLC, salt, for example, sodium chloride, sodium sulfate, potassium perchlorate, ammonium acetate, etc., preferably a volatile salt such as ammonium acetate is gradually increased to obtain each fraction. . The fraction itself or a sample obtained by concentrating and drying the fraction is subjected to enzymatic hydrolysis. As the enzyme, appropriate proteolytic enzymes such as various peptidases, trypsin, chymotrypsin and the like are used. At this time, enzyme hydrolysis can be performed online by connecting an enzyme column.
この溶液の一部を蛍光検出器付き逆相分配ＨＰＬＣに付し、蛍光標識体の溶出位置を確認する。次いで、この逆相ＨＰＬＣカラムの出口を質量分析計（どの様な質量分析計でも対応可能であるが、望ましくはエレクトロスプレー型質量分析計を用いる）に接続し、酵素水解物の蛍光標識フラグメント及び蛍光非標識フラグメントの質量分析（蛍光標識フラグメントは一回の質量分析、蛍光非標識フラグメントは親イオンを更に質量分析する）又は質量分析／質量分析（ＭＳ／ＭＳ）を行う。この際、蛍光検出器と質量分析計を直列に接続することも可能である。このようにして得られた各フラグメントのイオン分子量情報を、コンピューターに接続したタンパク質／ペプチドフラグメントデータベースと照合し、構造解析することにより、酵素水解以前のタンパク質／ペプチドの同定を行う。この場合、本発明では、タンパク質及び／又はペプチドフラグメント情報、及び蛍光試薬で標識したアミノ酸の情報を含んだデータベースを用いてデータベース照合を行う。 A part of this solution is subjected to reverse phase partition HPLC with a fluorescence detector to confirm the elution position of the fluorescent label. The reverse-phase HPLC column outlet is then connected to a mass spectrometer (any mass spectrometer can be used, but preferably an electrospray mass spectrometer), and the enzyme hydrolyzate fluorescently labeled fragment and Mass analysis of fluorescent unlabeled fragments (single mass analysis for fluorescently labeled fragments, mass analysis of parent ions further for fluorescent unlabeled fragments) or mass spectrometry / mass spectrometry (MS / MS) is performed. At this time, the fluorescence detector and the mass spectrometer can be connected in series. The ionic molecular weight information of each fragment thus obtained is collated with a protein / peptide fragment database connected to a computer, and structural analysis is performed to identify the protein / peptide before enzyme hydrolysis. In this case, in the present invention, database collation is performed using a database including protein and / or peptide fragment information and amino acid information labeled with a fluorescent reagent.
本発明では、発現タンパク質及び／又はペプチドを含む被験試料中のタンパク質及び／又はペプチドを蛍光誘導体化し、この蛍光誘導体をＨＰＬＣ／蛍光検出で分離し、蛍光ピークの強さを比較して標的発現タンパク質及び／又はペプチドの蛍光誘導体を分離し、得られた標的発現タンパク質及び／又はペプチドのピーク画分を酵素分解し、次いで、質量分析又はＭＳ／ＭＳ分析、データベース照合、構造解析により、上記タンパク質及び／又はペプチドを同定する。タンパク質及び／又はペプチドのアミノ基、チオール基、水酸基及びカルボキシル基などの機能性部分を誘導化するための多くの蛍光試薬が存在するので、本発明では、その目的に応じて、適当な試薬を任意に選択して使用することができる。後記する実施例に示されるように、例えば、Ｃｙｓ−含有タンパク質を誘導化するためには、チオール基に特異的な試薬である、Ａｍｍｏｎｉｕｍ ７−ｆｌｕｏｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉｚｏｌｅ−４−ｓｕｌｆｏｎａｔｅ（ＳＢＤ−Ｆ）を使用することができる。図１に、本発明の方法のプロセスの一例を模式的に示す。後記する実施例に示されるように、実際、このようにして、ラットに１０ｍｇのデキサメタゾンを投与して２日後のランゲルハンス島におけるＰａｎｃｒｅａｔｉｃ ｐｏｌｙｐｅｐｔｉｄｅ、プロインシュリン ２、７８ＫＤ Ｇｌｕｃｏｓｅ−ｒｅｇｕｌａｔｅｄ ｐｒｏｔｅｉｎ、プロテイン結合フォスファチジルアミン及びチオレドキシンが強く誘導されたことが示された。
In the present invention, the protein and / or peptide in the test sample containing the expressed protein and / or peptide is fluorescently derivatized, the fluorescent derivative is separated by HPLC / fluorescence detection, and the intensity of the fluorescent peak is compared to compare the target expressed protein And / or separating the fluorescent derivative of the peptide, enzymatically degrading the obtained target expressed protein and / or peptide peak fraction, and then analyzing the protein and the peptide by mass spectrometry or MS / MS analysis, database verification, and structural analysis. Identify peptides. There are many fluorescent reagents for derivatizing functional moieties such as amino groups, thiol groups, hydroxyl groups and carboxyl groups of proteins and / or peptides. In the present invention, an appropriate reagent is selected according to the purpose. It can be arbitrarily selected and used. As shown in the examples below, for example, to derivatize Cys-containing proteins, ammonia 7-fluor-2,1,3-benzoxazole-4-sulfonate, a reagent specific for thiol groups. (SBD-F) can be used. FIG. 1 schematically shows an example of the process of the method of the present invention. As shown in the examples below, in fact, in this way, rats were given 10 mg of dexamethasone and two days later Pancreatic polypeptide,
本発明の方法で重要な点は、各組織におけるタンパク質の量は、ＨＰＬＣ／蛍光検出で分離する前に、例えば、正常組織と非正常組織との組織間で定量、比較されるべきであることから、標的発現タンパク質を定量的に誘導体化することである。そのために、本発明では、適宜の界面活性剤が用いられるが、例えば、いくつかの界面活性剤についてＢＳＡにより検討したところ、ＣＨＡＰＳがｎ−Ｄｏｄｅｃｙｌ−β−Ｄ−ｍａｌｔｏｐｙｒａｎｏｓｉｄｅと比べて高い強度を示した（図２参照）。本発明では、ｐＨ、温度、反応時間及び誘導体化反応の添加剤等について、標的発現タンパク質及び／又はペプチドに応じて、最適条件を設定することで定量的な蛍光誘導体化が可能である。本発明では、これらの条件は、発現タンパク質／ペプチドの種類、分析目的等に応じて、適宜設定することができる。本発明の方法により、試験タンパク質／ペプチドのクロマトグラムは単一の蛍光ピークを示した（図３参照）。本発明の方法において、タンパク質及び／又はペプチドの検出限界は、０．２−６．０ｆｍｏｌであり、最適条件下での１０−１０００ｆｍｏｌの範囲で良好な直線（γ＞０．９９９４）の測定曲線が得られ（表１参照）、検出性能は、従来法に比べて、顕著であることが分かる。表１に、蛍光検出／ＨＰＬＣによる各種タンパク質及び／又はペプチドの検出限界を示した。 The important point in the method of the present invention is that the amount of protein in each tissue should be quantified and compared, for example, between normal and non-normal tissues before separation by HPLC / fluorescence detection. From this, the target expression protein is quantitatively derivatized. Therefore, in the present invention, an appropriate surfactant is used. For example, when several surfactants were examined by BSA, CHAPS showed higher strength than n-Dodecyl-β-D-maltopyranoside. (See FIG. 2). In the present invention, quantitative fluorescence derivatization is possible by setting optimum conditions for pH, temperature, reaction time, derivatization reaction additives, and the like according to the target expressed protein and / or peptide. In the present invention, these conditions can be appropriately set according to the type of expressed protein / peptide, the purpose of analysis, and the like. According to the method of the present invention, the chromatogram of the test protein / peptide showed a single fluorescence peak (see FIG. 3). In the method of the present invention, the detection limit of the protein and / or peptide is 0.2-6.0 fmol, and a measurement curve with a good straight line (γ> 0.9994) in the range of 10-1000 fmol under the optimum conditions. (See Table 1), and it can be seen that the detection performance is remarkable as compared with the conventional method. Table 1 shows detection limits of various proteins and / or peptides by fluorescence detection / HPLC.
更に、本発明では、上記方法に使用する微量の発現タンパク質及び／又はペプチド検出・分離・同定システムとして、被験試料のタンパク質及び／又はペプチドを蛍光試薬で標識するための第一反応器、蛍光試薬で標識した蛍光誘導体を蛍光分画するための１次元又は２次元の蛍光検出器付きＨＰＬＣ、蛍光分画を酵素水解するための第二反応器、酵素水解物の蛍光標識フラグメントを蛍光検出するための第二段階の蛍光検出器付きＨＰＬＣ、及び蛍光試薬で標識したアミノ酸の情報を含んだデータベースを搭載した構造解析装置の１種又は２種以上を構成要素として含む上記検出・分離・同定システムが用いられる。この場合、上記第一反応器、２次元の蛍光検出器付きＨＰＬＣ、第二反応器、第二段階の蛍光検出器付きＨＰＬＣを直列に配置することができる。これらの装置は、その使用目的に応じて、適宜の容量、形態に任意に設計することができる。 Furthermore, in the present invention, as a trace amount expressed protein and / or peptide detection / separation / identification system used in the above method, a first reactor for labeling a protein and / or peptide of a test sample with a fluorescent reagent, a fluorescent reagent HPLC with a one-dimensional or two-dimensional fluorescence detector for fluorescent fractionation of fluorescent derivatives labeled with, a second reactor for enzymatic hydrolysis of the fluorescent fraction, and fluorescence detection of fluorescently labeled fragments of the enzymatic hydrolyzate The above-mentioned detection / separation / identification system comprising, as a constituent element, one or more of a structural analysis apparatus equipped with a second stage HPLC equipped with a fluorescence detector and a database containing amino acid information labeled with a fluorescent reagent Used. In this case, the first reactor, the HPLC with the two-dimensional fluorescence detector, the second reactor, and the HPLC with the second stage fluorescence detector can be arranged in series. These devices can be arbitrarily designed to have an appropriate capacity and form according to the purpose of use.
本発明は、被験試料中のタンパク質及び／又はペプチドを、蛍光誘導体化試薬として、前記一般式（１）〔式中、Ｘは、ハロゲン、Ｙは、Ｏ、Ｓｅ又はＳ、Ｒは、−ＮＨ２、又は−ＮＨＲ′（但し、Ｒ′はＮ置換アルキル）を示す。〕で表わされる化合物、又は前記一般式（２）（式中、Ｘは、ハロゲン、Ｙは、Ｓｅ又はＳを示す。）で表わされる化合物、を用いて、蛍光誘導体とすることができる。更に、本発明は、これらの化合物のいずれか１種を有効成分とする新規蛍光誘導体化試薬を提供することができる。 In the present invention, a protein and / or peptide in a test sample is used as a fluorescent derivatization reagent, and the above general formula (1) [wherein, X is halogen, Y is O, Se or S, R is —NH 2 or -NHR '(where R' is an N-substituted alkyl). ] Or a compound represented by the general formula (2) (wherein X represents halogen, Y represents Se or S) can be used as a fluorescent derivative. Furthermore, the present invention can provide a novel fluorescent derivatization reagent containing any one of these compounds as an active ingredient.
これらの化合物の具体例としては、好適には、例えば、以下の例があげられるが、これらに制限されるものではなく、これらと同等ないし類似の化合物であれば同様に使用することができる。本発明の化合物は、後記する実施例に具体的に記載した方法と同様にして容易に合成することができる。
（１）ＤＡＡＢＤ−Ｃｌ[4-(dimethylaminoethyl aminosulfonyl)-7-chloro-2,
1, 3-benzoxadiazole]
（２）ＴＡＡＢＤ−Ｃｌ（7-chloro-2,
1, 3-benzoxadiazole-4-sulfonylaminoethyl rimethylammonium
chloride）
（３）ＤＡＡＢＤ−Ｆ[4-(dimethylaminoethyl aminosulfonyl)-7-fluoro-2,
1, 3-benzoxadiazole]
（４）ＴＡＡＢＤ−Ｆ（7-fluoro-2,
1, 3-benzoxadiazole-4-sulfonylaminoethyl trimethylammonium
chloride）
（５）ＤＡＡＢＳｅＤ−Ｃｌ[4-(dimethylaminoethyl aminosulfonyl)-7-chloro-2,
1, 3-benzoselenadiazole]
（６）ＴＡＡＢＳｅＤ−Ｃｌ（7-chloro-2,
1, 3-benzoselenadiazole-4-sulfonylaminoethyl trimethylammonium
chloride）
（７）ＤＡＡＢＳｅＤ−Ｆ[4-(dimethylaminoethyl aminosulfonyl)-7-fluoro-2,
1, 3- benzoselenadiazole]
（８）ＴＡＡＢＳｅＤ−Ｆ（7-fluoro-2,
1, 3-benzoselenadiazole-4-sulfonylaminoethyl trimethylammonium
chloride）
（９）ＤＡＡＢＴｈＤ−Ｃｌ[4-(dimethylaminoethyl aminosulfonyl)-7-chloro-2,
1, 3-benzothiadiazole]
（１０）ＴＡＡＢＴｈＤ−Ｃｌ（7-chloro-2,
1, 3-benzothiadiazole-4-sulfonylaminoethyl trimethylammonium
chloride）
（１１）ＤＡＡＢＴｈＤ−Ｆ[4-(dimethylaminoethyl aminosulfonyl)-7-fluoro-2,
1, 3-benzothiadiazole]
（１２）ＴＡＡＢＴｈＤ−Ｆ（7-fluoro-2,
1, 3-benzothiadiazole-4-sulfonylaminoethyl trimethylammonium
chloride）
Preferable examples of these compounds include, but are not limited to, the following examples, and any equivalent or similar compounds can be used in the same manner. The compounds of the present invention can be easily synthesized in the same manner as specifically described in the examples described later.
(1) DAABD-Cl [4- (dimethylaminoethyl aminosulfonyl) -7-chloro-2,
1, 3-benzoxadiazole]
(2) TAABD-Cl (7-chloro-2,
1, 3-benzoxadiazole-4-sulfonylaminoethyl rimethylammonium
chloride)
(3) DAABD-F [4- (dimethylaminoethyl aminosulfonyl) -7-fluoro-2,
1, 3-benzoxadiazole]
(4) TAABD-F (7-fluoro-2,
1, 3-benzoxadiazole-4-sulfonylaminoethyl trimethylammonium
chloride)
(5) DAABSeD-Cl [4- (dimethylaminoethyl aminosulfonyl) -7-chloro-2,
1,3-benzoselenadiazole]
(6) TAABSeD-Cl (7-chloro-2,
1, 3-benzoselenadiazole-4-sulfonylaminoethyl trimethylammonium
chloride)
(7) DAABSeD-F [4- (dimethylaminoethyl aminosulfonyl) -7-fluoro-2,
1,3-benzoselenadiazole]
(8) TAABSeD-F (7-fluoro-2,
1, 3-benzoselenadiazole-4-sulfonylaminoethyl trimethylammonium
chloride)
(9) DAABThD-Cl [4- (dimethylaminoethyl aminosulfonyl) -7-chloro-2,
1,3-benzothiadiazole]
(10) TAABThD-Cl (7-chloro-2,
1, 3-benzothiadiazole-4-sulfonylaminoethyl trimethylammonium
chloride)
(11) DAABThD-F [4- (dimethylaminoethyl aminosulfonyl) -7-fluoro-2,
1,3-benzothiadiazole]
(12) TAABThD-F (7-fluoro-2,
1, 3-benzothiadiazole-4-sulfonylaminoethyl trimethylammonium
chloride)
本発明により、１）遺伝子を介して発現する発現タンパク質及び／又はペプチドを簡便な方法及び手段で、高感度に検出・分離・同定することができる、２）本発明の方法により、従来法では検出できなかった微量の発現タンパク質及び／又はペプチドを短時間で、感度良く検出・分離・同定することができる、３）また、上記検出・分離・同定方法に使用する微量の発現タンパク質及び／又はペプチドの微量検出・分離・同定システムを提供することができる、４）本発明は、プロテオームのプラットフォーム技術を提供するものとして有用である、という効果が奏される。 According to the present invention, 1) an expressed protein and / or peptide expressed via a gene can be detected / separated / identified with high sensitivity by a simple method and means. 2) According to the method of the present invention, A small amount of expressed protein and / or peptide that could not be detected can be detected, separated and identified with high sensitivity in a short time. 3) Also, a small amount of expressed protein and / or used in the above detection, separation and identification method It is possible to provide a trace amount detection / separation / identification system for peptides. 4) The present invention has an effect of being useful as a platform technology for proteome.
次に、実施例に基づいて本発明を具体的に説明するが、本発明は、以下の実施例によって何ら限定されるものではない。 EXAMPLES Next, although this invention is demonstrated concretely based on an Example, this invention is not limited at all by the following Examples.
ラット膵ランゲルハンス島（ラ島）中チオール含有タンパク質／ペプチドの分離・同定（１）ラ島中チオール含有タンパク質／ペプチドの蛍光誘導体化
ラ島に０．１Ｍホウ酸緩衝液（ｐＨ９．０）に溶解した６Ｍ塩酸グアニジン溶液５０μｌを加えて可溶化した。これに６Ｍ塩酸グアニジン溶液に溶解した１７．５ｍＭＴＣＥＰ、１７．５ｍＭＳＢＤ−Ｆ、１０ｍＭＥＤＴＡ及び５０ｍＭＣＨＡＰＳをそれぞれ５０μｌずつ加え、混合した。この溶液を４０℃にて３時間反応させることにより蛍光誘導体化を行った。
（２）イオン交換ＨＰＬＣによる１次分離
上記の反応溶液をイオン交換カラムに付し、ＮａＣｌのグラジエント（０、０．０４、０．０８、０．１２及び０．３Ｍ）により蛍光タンパク質／ペプチドを溶出させ、５つのフラクションに分離した。なお、蛍光タンパク質／ペプチドの検出はＳＢＤ骨格の蛍光により行った。ＨＰＬＣ条件を以下に示す。
Isolation and identification of thiol-containing proteins / peptides in rat pancreatic islets (Ra island) (1) Fluorescence derivatization of thiol-containing proteins / peptides in Ra islands Dissolved in 0.1 M borate buffer (pH 9.0) in
(2) Primary separation by ion exchange HPLC The above reaction solution is applied to an ion exchange column, and fluorescent protein / peptide is separated by NaCl gradient (0, 0.04, 0.08, 0.12 and 0.3M). Elute and separate into 5 fractions. The fluorescent protein / peptide was detected by fluorescence of the SBD skeleton. The HPLC conditions are shown below.
（ＨＰＬＣ条件）
カラム：ＴＳＫｇｅｌ ＤＥＡＥ−５ＰＷ ７．５×７５ｍｍ（東ソー（株））
ガードカラム：Ｃ８−３００−Ｓ ５４．０×１０ｍｍ（ＹＭＣ（株））
移動相：段階溶離〔０−５分：Ｃ１００％、５−１５分：Ａ１００％、１５−２５分：Ａ８７％Ｂ１３％、２５−３５分Ａ７３％Ｂ２７％、３５−４５分：Ａ６０％Ｂ４０％、４５−５５分：Ｂ１００％〕
Ａ：５ｍＭトリス塩酸緩衝液（ｐＨ８．０）／アセトニトリル（５０：５０）
Ｂ：５ｍＭトリス塩酸緩衝液（ｐＨ８．０）／アセトニトリル（５０：５０） （０．３ＭＮａＣｌ含有）
Ｃ：５ｍＭトリス塩酸緩衝液（ｐＨ８．０）
カラム温度：室温（約２５℃））
流速：０．５ｍｌ／ｍｉｎ
検出：Ｅｘ３８０ｎｍ、 Ｅｍ５０５ｎｍ
注入量：２００μｌ
(HPLC conditions)
Column: TSKgel DEAE-5PW 7.5 × 75 mm (Tosoh Corporation)
Guard column: C8-300-S 54.0 × 10 mm (YMC Corporation)
Mobile phase: step elution [0-5 min: C100%, 5-15 min: A100%, 15-25 min: A87% B13%, 25-35 min A73% B27%, 35-45 min: A60% B40% 45-55 minutes: B100%]
A: 5 mM Tris-HCl buffer (pH 8.0) / acetonitrile (50:50)
B: 5 mM Tris-HCl buffer (pH 8.0) / acetonitrile (50:50) (containing 0.3 M NaCl)
C: 5 mM Tris-HCl buffer (pH 8.0)
Column temperature: room temperature (about 25 ° C)
Flow rate: 0.5 ml / min
Detection: Ex380nm, Em505nm
Injection volume: 200 μl
（３）逆相ＨＰＬＣによる２次分離
上記の各画分を濃縮し、アセトニトリルを蒸発させた後、逆相カラムに付し、アセトニトリルの勾配溶離によりタンパク質／ペプチドをカラムから溶出させた。なお、タンパク質／ペプチドの検出はＳＢＤ骨格の蛍光によりモニターした。ＨＰＬＣ条件を以下に示す。
(3) Secondary separation by reverse phase HPLC Each of the above fractions was concentrated and the acetonitrile was evaporated, and then applied to a reverse phase column, and the protein / peptide was eluted from the column by gradient elution of acetonitrile. Protein / peptide detection was monitored by fluorescence of the SBD skeleton. The HPLC conditions are shown below.
（ＨＰＬＣ条件）
カラム：カプセルパックＣ８ ＳＧ３００ ２．０×１００ｍｍ（（株）資生堂）
移動相：勾配溶離（０→６０分：Ｂ４０％→１００％）
Ａ：０．０５％トリフルオロ酢酸
Ｂ：０．０５％トリフルオロ酢酸／アセトニトリル（４０：６０）
カラム温度：室温（約２５℃）
流速：０．２ｍｌ／ｍｉｎ
検出：Ｅｘ３８０ｎｍ、 Ｅｍ５０５ｎｍ
注入量：５０μｌ
(HPLC conditions)
Column: Capsule pack C8 SG300 2.0 × 100 mm (Shiseido Co., Ltd.)
Mobile phase: gradient elution (0 → 60 min: B40% → 100%)
A: 0.05% trifluoroacetic acid B: 0.05% trifluoroacetic acid / acetonitrile (40:60)
Column temperature: Room temperature (about 25 ° C)
Flow rate: 0.2 ml / min
Detection: Ex380nm, Em505nm
Injection volume: 50 μl
（４）酵素処理
採取した画分をそれぞれのチューブに０．５Ｍ炭酸水素アンモニウム溶液５μｌを加えてトリフルオロ酢酸を中和後、濃縮してアセトニトリルを蒸発させた。残渣（約８０μｌ）に２０μｇ／ｍｌトリプシン（プロメガ）及び１０ｍＭ塩化カルシウムをそれぞれ１０μｌずつ添加した。これを３７℃で２時間インキュベートし、ＭＳ／ＭＳ測定用の試料とした。
(4) Enzyme treatment The collected fraction was neutralized with trifluoroacetic acid by adding 5 μl of 0.5 M ammonium hydrogen carbonate solution to each tube, and then concentrated to evaporate acetonitrile. 10 μl each of 20 μg / ml trypsin (Promega) and 10 mM calcium chloride was added to the residue (about 80 μl). This was incubated at 37 ° C. for 2 hours to obtain a sample for MS / MS measurement.
（５）ＭＳ／ＭＳ測定によるタンパク質／ペプチドの同定
上記の試料を逆相カラムＨＰＬＣに付し、エレクトロスプレー法によるＭＳ／ＭＳ測定を行った。ＨＰＬＣ条件を以下に示す。なお、タンパク質／ペプチドの同定はデータベースにＮＣＢＩ、サーチエンジンにＭＡＳＣＯＴを使用した。
(5) Identification of protein / peptide by MS / MS measurement The above sample was subjected to reverse phase column HPLC, and MS / MS measurement by electrospray method was performed. The HPLC conditions are shown below. For identification of proteins / peptides, NCBI was used as a database and MASCOT was used as a search engine.
（ＨＰＬＣ条件）
カラム：Ｃａｄｅｎｚａ ＴＣ−Ｃ１８ ２．０ ×１００ｍｍ（Ｉｍｔａｃｔ（株））
移動相：勾配溶離（０→３０分：Ｂ２０％→１００％）
Ａ：０．１％ギ酸
Ｂ：０．１％ギ酸／アセトニトリル（５０：５０）
カラム温度：室温（約２５℃）
流速：０．２ｍｌ／ｍｉｎ
測定モード：ｐｏｓｉｔｉｖｅ
測定範囲：５００−３０００ｍ／ｚ
注入量：５０μｌ
(HPLC conditions)
Column: Cadenza TC-C18 2.0 × 100 mm (Imactact)
Mobile phase: gradient elution (0 → 30 min: B20% → 100%)
A: 0.1% formic acid B: 0.1% formic acid / acetonitrile (50:50)
Column temperature: Room temperature (about 25 ° C)
Flow rate: 0.2 ml / min
Measurement mode: positive
Measurement range: 500-3000 m / z
Injection volume: 50 μl
上記の方法により、約１３０のタンパク質／ペプチドのピークが分離できた。
そのうち、約５０のタンパク質／ペプチドが同定できた（表２、図６参照）。
About 130 protein / peptide peaks could be separated by the above method.
Among them, about 50 proteins / peptides were identified (see Table 2 and FIG. 6).
ＳＢＤ−Ｆで誘導体化したＢＳＡを、図１に示されるプロセスにより、トリプシンで酵素水解し、得られたペプチド混合物を逆相液体クロマトグラフィー（ＲＰＬＣ）で分離し、蛍光検出器で検出した。次いで、各ペプチドをＥＳＩイオンタップ質量分析計によるＭＳ／ＭＳ分析に供した。原理的には、トリプシン分解により、ＢＳＡは、４個のアミノ酸残基以上の２５のシステイン含有ペプチド及
び３５のシステイン非含有ペプチドが生成されるが、本実施例では、２７以上の蛍光ペプチドが蛍光検出され、定量的に誘導体化が行われた（図４、Ａ）。
BSA derivatized with SBD-F was enzymatically hydrolyzed with trypsin according to the process shown in FIG. 1, and the resulting peptide mixture was separated by reverse phase liquid chromatography (RPLC) and detected with a fluorescence detector. Each peptide was then subjected to MS / MS analysis using an ESI ion tap mass spectrometer. In principle, by trypsin degradation, BSA produces 25 cysteine-containing peptides with 4 or more amino acid residues and 35 cysteine-free peptides. In this example, 27 or more fluorescent peptides are fluorescent. Detected and quantitatively derivatized (FIG. 4, A).
また、１１のシステイン含有ペプチド及び１７の非システイン含有ペプチドがマスクロマトグラフィーで検出された（図４、Ｂ）。 図５に、（Ｍ＋２Ｈ）2+プレカーサー、ｍ／ｚ＝８７３．４（図４で矢印で示した）から得たＭＳ／ＭＳスペクトルを示す。
全てのペプチドフラグメントのＣＩＤスペクトルにより、確率的プロテイン同定法に基づくＭＡＳＣＯＴによるデータベース照合を行い、予測通り、完全にＢＳＡとしてタンパク質を同定した（スコア：３９）。
In addition, 11 cysteine-containing peptides and 17 non-cysteine-containing peptides were detected by mass chromatography (FIG. 4, B). FIG. 5 shows the MS / MS spectrum obtained from (M + 2H) 2+ precursor, m / z = 873.4 (indicated by arrows in FIG. 4).
Based on the CID spectra of all peptide fragments, MASCOT database verification based on the probabilistic protein identification method was performed, and as expected, the protein was completely identified as BSA (score: 39).
デキサメタゾン（Ｄｅｘ）投与及び非投与のラット膵臓について試験した。
Ｄｅｘは、肝臓のグルコース産生の増加とインシュリン耐性の誘導により主なヒト糖尿病であるタイプ２の糖尿病を誘発する。実際に、Ｄｅｘ処理の２４時間後に、血中グルコースレベルは２０９．８ｍｇ／ｄＬに達し、処理前の値の１１８．３ｍｇ／ｄＬより著しく高い（ｐ＜０．０５）。本実施例では、２日間Ｄｅｘで処理又は未処理のラット膵臓からランゲルハンス島（６０島）を採取し、Ｓ
ＢＤ−Ｆで誘導体化した。本発明の方法を生物試料に適用するための重要な態様は、タンパク質混合物からＨＰＬＣで発現タンパク質を分離することである。本実施例では、蛍光タンパク質は、まず、ＳＢＤ−Ｆにより生成した多くのマイナス電荷と酸性アミノ酸部分に基づいてイオン交換クロマトグラフィー（ＩＥＣ）で分離された。
Rat pancreas with and without dexamethasone (Dex) was tested.
Dex induces
Derivatized with BD-F. An important aspect for applying the method of the invention to biological samples is to separate the expressed protein by HPLC from the protein mixture. In this example, fluorescent proteins were first separated by ion exchange chromatography (IEC) based on many negative charges and acidic amino acid moieties generated by SBD-F.
ＩＥＣは、塩化ナトリウムの段階溶離（０、０．０４、０．０８、０．１２、及び０．３Ｍ ＮａＣｌ）により行い、蛍光タンパク質混合物を５つの画分として得た。次いで、各画分は、更に、それらの疎水性に基づいて、逆相液体クロマトグラフィー（ＲＰＬＣ）により分離された。この実験でのピークの容量（ｎ＝Ｌ／（４σ）、但し、Ｌは分析のトータル時間及び４σはピーク幅、としてのＨＰＬＣの性能の理論値）は、各ＲＰＬＣフラクションにつき４０と計算され、ＩＥＣ−ＲＰＬＣ法の５つのステップのピーク容量は、約２００であった。本実施例では、各ＲＰＬＣサイクルで約３−５０ピーク、合計で１２９ピークであった（図６）。 IEC was performed by step elution of sodium chloride (0, 0.04, 0.08, 0.12, and 0.3 M NaCl) to give a fluorescent protein mixture as 5 fractions. Each fraction was then further separated by reverse phase liquid chromatography (RPLC) based on their hydrophobicity. The peak volume in this experiment (n = L / (4σ), where L is the total time of analysis and 4σ is the peak width, the theoretical value of HPLC performance) is calculated to be 40 for each RPLC fraction, The peak capacity of the five steps of the IEC-RPLC method was about 200. In this example, there were about 3-50 peaks in each RPLC cycle, totaling 129 peaks (FIG. 6).
微量タンパク質を検出するために、ＩＥＣの段階溶離ステップを増やしてピーク容量を増加させた。未処理及びＤｅｘ処理ラットから得られたＲＰＬＣクロマトグラムの全ての蛍光ピークを比較した。その結果、５本の蛍光ピークが１．８以上増加し、３本の蛍光ピークがＤｅｘ処理で約１．５倍減少したことが見出された（表２）。表２に、Ｄｅｘ投与２日後の発現タンパク質の変化を示した。これらのタンパク質（即ち、標的発現タンパク質）は、広い小孔を有するＲＰＬＣ（３０ｎｍ小孔径）で分離され、トリプシンで分解し、各ペプチド混合物とした。各ペプチド混合物は、通常の小孔を有するＲＰＬＣ（１０ｎｍ小孔径）に供し、ＭＳ／ＭＳ分析した。データベース照合により、Ｄｅｘ処理後２日で増加したピークは、各々、膵臓ポリペプチド、プロインシュリン２、７８ＫＤグルコース−調節タンパク質、ホスファチジルエタノールアミン結合タンパク質、及びチオレドキシンであり、減少したピークは、各々、プロテイン３１、ｄｎａｋ−タイプ分子キャペロンｈｓｐ７２−ｐｓｌ、及びインシュリンであった。
In order to detect trace proteins, the IEC step elution step was increased to increase the peak volume. All fluorescence peaks of RPLC chromatograms obtained from untreated and Dex-treated rats were compared. As a result, it was found that the five fluorescent peaks increased by 1.8 or more and the three fluorescent peaks decreased about 1.5 times by Dex treatment (Table 2). Table 2 shows changes in expressed
本実施例では、以下の化１の（１）及び（２）の反応式により、新規蛍光誘導体化試薬の合成を行った。 In this embodiment, by the reaction equation of the following Formula 1 (1) and (2), was synthesized novel fluorescent derivatization reagent.
（１）DAAB-Clの合成
4-chlorosulfonyl-7-chloro-2,1,3-benzoxadiazole(CBD-Cl)(126.53 mg)をCH3CN に溶解し、N,N-dimethylethylenediamineを滴下し、triethylamineを加えた。室温で約10分間攪拌後、反応液を減圧乾固した後、シリカゲルカラム(CH2Cl2)で精製し、4-(dimethylaminoethy laminosulfonyl)-7-chloro-2,1,3-benzoxadiazole(DAABD-Cl) （20.2 mg, 87.4
%)を得た。
得られた化合物の確認データを以下に示す。
1H-NMR (CD3OD) : 7.94 (1H, d,
J=7.5), 7.65 (1H, d, J=7.5), 3.06 (2H, t, J=6.7), 2.30 (2H, t, J=6.7), 2.02
(6H, s)。ESI-MS : m/z 305 (M+H)+
(1) Synthesis of DAAB-Cl
4-chlorosulfonyl-7-chloro-2,1,3-benzoxadiazole (CBD-Cl) (126.53 mg) was dissolved in CH 3 CN, N, N-dimethylethylenediamine was added dropwise, and triethylamine was added. After stirring at room temperature for about 10 minutes, the reaction solution was dried under reduced pressure and purified with a silica gel column (CH 2 Cl 2 ), and 4- (dimethylaminoethylaminosulfonyl) -7-chloro-2,1,3-benzoxadiazole (DAABD- Cl) (20.2 mg, 87.4
%).
Confirmation data of the obtained compound is shown below.
1H-NMR (CD 3 OD): 7.94 (1H, d,
J = 7.5), 7.65 (1H, d, J = 7.5), 3.06 (2H, t, J = 6.7), 2.30 (2H, t, J = 6.7), 2.02
(6H, s). ESI-MS: m / z 305 (M + H) +
（２）ＴＡＡＢＤ−Ｃｌの合成
４−ｃｈｌｏｒｏｓｕｌｆｏｎｙｌ−７−ｃｈｌｏｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ（ＣＢＤ−Ｃｌ）（１２６．５３ｍｇ）をＣＨ３ＣＮに溶解し、Ｈ２Ｏに溶かしたａｍｉｎｏｅｔｈｙｌ ｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍ ｃｈｌｏｒｉｄｅを滴下し、ｔｒｉｅｔｈｙｌａｍｉｎｅを加えた。室温で約２０分間攪拌後、反応液を減圧乾固した後、０．１％トリフルオロ酢酸（ＴＦＡ）に溶かし、ＯＤＳカラムを用いて分取し、画分には、ＳＢＤ−Ｃｌ（化２）が不純物として入っていたため、陰イオン交換カラムを用いて分取し、減圧乾固して、７−ｃｈｌｏｒｏ−２，１，３−ｂｅｎｚｏｘａｄｉａｚｏｌｅ−４−ｓｕｌｆｏｎｅａｍｉｎｏｅｔｈｙｌ ｔｒｉｍｅｔｈｙｌａｍｍｏｎｉｕｍ ｃｈｌｏｒｉｄｅ（ＴＡＡＢＤ−Ｃｌ）（１２７．２ｍｇ，５８．８％）を得た。
得られた化合物の確認データを以下に示す。
１Ｈ−ＮＭＲ（ＣＤ３ＯＤ）：８．０１（１Ｈ，ｄ，Ｊ＝７．３），７．６９（１Ｈ，ｄ，Ｊ＝７．３），３．４６−３．４８（４Ｈ，ｍ），３．１２（９Ｈ，ｓ）。ＥＳＩ−ＭＳ ：ｍ／ｚ ３１９（Ｍ）＋
(2) Synthesis of TAABD-Cl 4-chlorosulfonyl-7-chloro-2,1,3-benzazodiazole (CBD-Cl) (126.53 mg) was dissolved in CH 3 CN and dissolved in H 2 O, an aminoethyl trimethylammonium chloride Was added dropwise and triethylamine was added. After stirring for about 20 minutes at room temperature, after the reaction solution was evaporated to dryness under reduced pressure, dissolved in 0.1% trifluoroacetic acid (TFA), aliquoted using an ODS column, the fractions, SBD-Cl (formula 2 ) Was contained as an impurity, and therefore, it was collected using an anion exchange column, dried under reduced pressure, and 7-chloro-2,1,3-benzazodiazole-4-sulfaminoaminotrimethylammonium chloride (TAABD-Cl) (127. 2 mg, 58.8%).
Confirmation data of the obtained compound is shown below.
1H-NMR (CD 3 OD): 8.01 (1H, d, J = 7.3), 7.69 (1H, d, J = 7.3), 3.46-3.48 (4H, m ), 3.12 (9H, s). ESI-MS: m / z 319 (M) +
本実施例では、新規蛍光誘導体化試薬の反応性について検討した。
DAABD-C、TAABDD-ClのSBD-Fとの比較
１０μＭ還元型グルタチオン、システイン、ホモシステイン混合液100μＬとDAABD-Cl又はTAABD-Cl 100 μLを混合し、pH 9、40℃、10〜120分間反応させた。尚、各試薬は5 ｍM EDTA含む0.10 M ホウ酸緩衝液 (pH 9) に溶解した。0.1 % ぎ酸で反応停止後、生成物をＨＰＬＣを用いて測定した。
In this example, the reactivity of the novel fluorescent derivatization reagent was examined.
Comparison of DAABD-C and TAABDD-Cl with SBD-
図７に、蛍光誘導体化反応時間と蛍光強度との関係（左図：ＤＡＡＢＤ−Ｃｌ、右図： ＴＡＡＢＤ−Ｃｌ）を示す。
ＳＢＤ−Ｆ（化３）の場合、４０℃で誘導体化を行うと１２０分間の反応時間が必要であったが、ＤＡＡＢＤ−Ｃｌは１０〜２０分、ＴＡＡＢＤ−Ｃｌは２０〜３０分で反応が終了することがわかった。
したがって、反応時間はＤＡＡＢＤ−Ｃｌの場合は２０分、ＴＡＡＢＤ−Ｃｌの場合は３０分が好適である。
FIG. 7 shows the relationship between the fluorescence derivatization reaction time and the fluorescence intensity (left diagram: DAABD-Cl, right diagram: TAABD-Cl).
In the case of SBD-F (Chemical Formula 3 ), 120 minutes of reaction time was required when derivatization was performed at 40 ° C., but DAABD-Cl was reacted for 10 to 20 minutes, and TAABD-Cl was reacted for 20 to 30 minutes. I knew it would end.
Accordingly, the reaction time is preferably 20 minutes for DAABD-Cl and 30 minutes for TAABD-Cl.
（２）新規蛍光誘導体化試薬のMSでの感度
上記（1）で作成したサンプルをLC-MSにより検出し、蛍光誘導体化試薬でラベル化していないもの及びSBD-Fで誘導体化したものと、相対強度を比較した。
(2) Sensitivity in MS of novel fluorescent derivatization reagent The sample prepared in (1) above was detected by LC-MS, and the one not labeled with the fluorescent derivatization reagent and the one derivatized with SBD-F, The relative intensities were compared.
蛍光誘導体化試薬でラベル化していないcysteine、homocysteine、GSHの高さをそれぞれ１としたときの相対強度は表3の通りであった。これより、DAABD-Cl がMSでの感度が最も高いことがわかった。また、移動相が酸性であるので、DAABD化誘導体はプラスに荷電され水溶性であると考えられる。
Table 3 shows the relative intensities when the heights of cysteine, homocysteine, and GSH not labeled with a fluorescent derivatization reagent are each 1. From this, it was found that DAABD-
（１）TAABD-Clのペプチドへの応用
10μMの以下に示した4種類のペプチド標品、17.5 mM TAABD-Cl、10 mM EDTA、50 mM CHAPS（界面活性剤）、2.5 mM TCEP（還元剤）それぞれ50μLを混合し、pH 9、40℃で、30, 60, 90, 120 分間反応させた。尚、各試薬は6.0
M 塩酸グアニジン（タンパク変性剤）を含む0.10
M ホウ酸緩衝液 (pH9) に溶解した。生成したTAABD化ペプチドをHPLCを用いて測定した。
1. vasopressin
2. oxytocin
3. somatostatin
4. amylin (rat)
図８に TAABD-Clとの反応時間と蛍光強度との関係を示す。
図８より、反応時間が60分までは生成量が増加した。反応停止後は氷冷下で保存し、-20℃にて保存すると、48時間はほとんど分解しなかった。
(1) Application of TAABD-Cl to peptides
10 μM of the following four peptide preparations, 17.5 mM TAABD-Cl, 10 mM EDTA, 50 mM CHAPS (surfactant), 2.5 mM TCEP (reducing agent) 50 μL each, mixed at
M 0.10 containing guanidine hydrochloride (protein denaturant)
Dissolved in M borate buffer (pH 9). The produced TAABD peptide was measured using HPLC.
1. vasopressin
2. Oxytocin
3. somatostatin
4. amylin (rat)
FIG. 8 shows the relationship between TAABD-Cl reaction time and fluorescence intensity.
From FIG. 8, the amount of production increased until the reaction time was 60 minutes. After stopping the reaction, it was stored under ice-cooling, and when it was stored at -20 ° C, it hardly decomposed for 48 hours.
（２）DAABD化ペプチド・タンパクの検出限界
表４に示した１０種類のペプチド・タンパク標品10
μM混合液、2.5 mM TCEP、17.5 mM DAABD-Cl、10 mM EDTA、50 mM CHAPSそれぞれ50μＬを混合し、pH 9、４０℃で、30分間反応させた。尚、各試薬は6.0
M 塩酸グアニジンを含む0.10 M ホウ酸緩衝液 (pH9) に溶解した。生成したDAABD化ペプチド・タンパクはHPLCを用いて測定し、蛍光検出の検出限界をSBD-Fと比較した。
(2) Detection limit of DAABD peptide /
50 μL each of μM mixed solution, 2.5 mM TCEP, 17.5 mM DAABD-Cl, 10 mM EDTA, 50 mM CHAPS were mixed and reacted at
Dissolved in 0.10 M borate buffer (pH 9) containing M guanidine hydrochloride. The generated DAABD peptide / protein was measured using HPLC, and the detection limit of fluorescence detection was compared with that of SBD-F.
（３）DAABD化ペプチド・タンパクの同定
上記（２）で誘導体化した物質のうち、vasopressin、oxytocin、somatostatin、calcitonin、amylinはLC-MSにより同定できた。それらの分子量を以下に示す。
m/z
541.8 (M+3H)3+ [DAABD-vasopressin]
516.0 (M+3H)3+
[DAABD-oxytocin]
726.6 (M+3H)3+
[DAABD-somatostatin]
989.9 (M+4H)4+
[DAABD-calcitonin]
892.8 (M+5H)5+
[DAABD-amylin]
(3) Identification of DAABD peptide / protein Among the substances derivatized in (2) above, vasopressin, oxytocin, somatostatin, calitonin and amylin could be identified by LC-MS. Their molecular weights are shown below.
m / z
541.8 (M + 3H) 3+ [DAABD-vasopressin]
516.0 (M + 3H) 3+
[DAABD-oxytocin]
726.6 (M + 3H) 3+
[DAABD-somatostatin]
989.9 (M + 4H) 4+
[DAABD-calcitonin]
892.8 (M + 5H) 5+
[DAABD-amylin]
これら全ての分子量は、それぞれのペプチドの２つのシステイン残基にDAABDが付加したとしたときの分子量であり、多価イオンピークの検出結果よりDAABD-Clによる誘導体化において、これらのペプチドのシステイン残基間のS-S結合が還元され、二つのチオール基両方に試薬が反応したことがわかった。
また、タンパク質の場合は酵素によってペプチドに分解する必要があるため、酵素トリプシンで消化し、LC-MS/MS検出及びMASCOTによるデータベース検索を行って同定を試みた結果、システインを含まないペプチドのアミノ酸配列を同定し、タンパク質を同定することができた。
All of these molecular weights are the molecular weights when DAABD is added to the two cysteine residues of each peptide. From the detection result of the polyvalent ion peak, derivatization with DAABD-Cl reveals the cysteine residues of these peptides. It was found that the SS bond between the groups was reduced and the reagent reacted to both two thiol groups.
In addition, in the case of protein, it is necessary to break down into peptides by an enzyme, and as a result of digestion with the enzyme trypsin and identification by performing a database search with LC-MS / MS detection and MASCOT, amino acids of peptides that do not contain cysteine The sequence was identified and the protein could be identified.
（ＳＢＳｅＤ−Ｆの合成）
2-fluoroacetanilideを硝酸で処理して、1-acetylamino-2-nitro-6-fluorobenzeneとし、これを脱アルミ化して、2-fluoro-6-nitroanillineとし、次いで、パラジウム担持炭素触媒を用いて水素化して、3-fluoro-o-phenylenediamineを得た。
Selenium dioxideエタノール加熱溶液を、3-fluoro-o-phenylenediamine (60 mg,
0.48 mmol)のエタノール加熱溶液に加え、混合物を３０分加熱した。これを、シリカゲルカラムによるクロマトグラフィーに供し、溶離液のジクロロメタンで溶出し、4-fluoro-2, 1, 3-benzoselenadiazoleを白色粉末（８８mg）として得た。得られた化合物の確認データを以下に示す。
mp. 129℃、NMR (methanol-d4): δＨ７．５５（１H, d, J=9.2）、７．４１(１Ｈ, ｍ）、７．０６（１Ｈ, ｍ）、ＥＳＩ−ＭＳ：ｍ／ｚ２０２．８［（Ｍ＋Ｈ）］。
(Synthesis of SBSeD-F)
2-fluoroacetanilide is treated with nitric acid to give 1-acetylamino-2-nitro-6-fluorobenzene, which is dealuminated to 2-fluoro-6-nitroanilline, and then hydrogenated using a palladium-supported carbon catalyst. Thus, 3-fluoro-o-phenylenediamine was obtained.
Selenium dioxide ethanol solution was added to 3-fluoro-o-phenylenediamine (60 mg,
0.48 mmol) in ethanol, and the mixture was heated for 30 minutes. This was subjected to chromatography on a silica gel column and eluted with dichloromethane as an eluent to give 4-fluoro-2,1,3-benzoselenadiazole as a white powder (88 mg). Confirmation data of the obtained compound is shown below.
. mp 129 ℃, NMR (methanol -d 4): δ H 7.55 (1H, d, J = 9.2), 7.41 (1H, m), 7.06 (1H, m), ESI-MS: m / z 202.8 [(M + H)].
このようにして得た4-fluoro-2, 1,
3-benzoselenadiazoleをfuming sulfuric
acid (60%)に溶かし、１３０℃で３時間還流した。この溶液を冷却し、冷水（３０ｍｌ）に注ぎ、２８％ ammonium hydroxideで中和した。この中性溶液にエタノール１００ｍｌを加え、濾過物を減圧乾固させた。残渣を水（１．０ｍｌ）に溶解し、更に、以下のＨＰＬＣで精製した。即ち、残渣の１００μｌをＨＰＬＣ分離に供した。ＳＢＳｅＤ−Ｆに相当するフラクションを集め、減圧して白色粉末（５０ｍｇ）を得た。得られた化合物の確認データを以下に示す。
ｍ．P． > 300℃、NMR (methanol-d4): δＨ７．９７（１H, d d, J=７．６, J=５．４）、７．１１(１Ｈ, d d，J=７．６, J=１０．１）、ＥＳＩ−ＭＳ：ｍ／ｚ２８０．８［（Ｍ−Ｈ）］。
4-fluoro-2, 1, 1, obtained in this way
3-benzoselenadiazole fuming sulfuric acid
It was dissolved in acid (60%) and refluxed at 130 ° C. for 3 hours. The solution was cooled, poured into cold water (30 ml) and neutralized with 28% ammonium hydroxide. 100 ml of ethanol was added to this neutral solution, and the filtrate was dried under reduced pressure. The residue was dissolved in water (1.0 ml) and further purified by the following HPLC. That is, 100 μl of the residue was subjected to HPLC separation. Fractions corresponding to SBSeD-F were collected and decompressed to give a white powder (50 mg). Confirmation data of the obtained compound is shown below.
m. P. > 300 ° C., NMR (methanol-d 4 ): δ H 7.97 (1H, dd, J = 7.6, J = 5.4), 7.11 (1H, dd, J = 7.6, J = 10.1), ESI-MS: m / z 280.8 [(M-H)].
（ＳＢＴｈＤ−Ｆの合成）
Ｎ−thionylaniline (0.49 g, 3.5 mmol)を3-fluoro-o-phenylenediamine（200mg, 1.6mmol）トルエン（2 ml）溶液に加えた。反応混合物を１００−１２０℃で４時間加熱し、溶媒を濾別した後、残渣をジクロロメタンに溶かし、溶液を１０％ＨＣｌ溶液及び水で各々洗浄した。有機相を乾燥し、減圧乾固させた。これをシリカゲルによるクロマトグラフィーに供し、溶離液のクロロホルムで溶出し、4-fluoro-2, 1, 3-benzothiadiazoleを淡黄色油として得た。得られた化合物の確認データを以下に示す。
NMR(methanol-d4): δH７．６９（１H, d, J=８．９）、７．５０(１Ｈ, ｍ）、７．２０（１Ｈ，ｍ）、ＥＳＩ−ＭＳ：ｍ／ｚ１５４．９［（Ｍ＋Ｈ）］。
(Synthesis of SBThD-F)
N-thionylaniline (0.49 g, 3.5 mmol) was added to a solution of 3-fluoro-o-phenylenediamine (200 mg, 1.6 mmol) in toluene (2 ml). The reaction mixture was heated at 100-120 ° C. for 4 hours, the solvent was filtered off, the residue was dissolved in dichloromethane, and the solution was washed with 10% HCl solution and water, respectively. The organic phase was dried and evaporated to dryness. This was subjected to chromatography on silica gel and eluted with chloroform as eluent to give 4-fluoro-2, 1, 3-benzothiadiazole as a pale yellow oil. Confirmation data of the obtained compound is shown below.
NMR (methanol-d 4 ): δ H 7.69 (1H, d, J = 8.9), 7.50 (1H, m), 7.20 (1H, m), ESI-MS: m / z 154 .9 [(M + H)].
このようにして得た４−ｆｌｕｏｒｏ−２，１，３−ｂｅｎｚｏｔｈｉａｄｉａｚｏｌｅ（３０ｍｌ）をｆｕｍｉｎｇ ｓｕｌｆｕｒｉｃ ａｃｉｄ（６０％）に溶かし、１３０℃で３時間還流した。次いで、この溶液を冷却し、ゆっくり冷水（３０ｍｌ）に注ぎ、２８％ ａｍｍｏｎｉｕｍ ｈｙｄｒｏｘｉｄｅで中和した。中性溶液にエタノール１００ｍｌを加え、得られた濾過物を減圧乾固した。残渣を水（１．０ｍｌ）に溶かし、更に、以下の条件でＨＰＬＣで精製した。ＳＢＴｈＤ−Ｆに相当するフラクションを集め、減圧し、白色粉末（２５ｍｇ）を得た。得られた化合物の確認データを以下に示す。
ｄｅｃｏｍｐ．２６５℃、ＮＭＲ（ｍｅｔｈａｎｏｌ−ｄ４）：δＨ８．０６（１Ｈ，ｄ ｄ，Ｊ＝７．９，Ｊ＝４．９）、７．１１（１Ｈ，ｄ ｄ，Ｊ＝７．９，Ｊ＝９．８）、ＥＳＩ−ＭＳ：ｍ／ｚ２３２．８［（Ｍ−Ｈ）］。
上記方法により合成したＳＢＳｅＤ−Ｆ及びＳＢＴｈＤ−Ｆを下記の化４に示す。
The 4-fluoro-2,1,3-benzothiazole (30 ml) thus obtained was dissolved in fuming sulfur acid (60%) and refluxed at 130 ° C. for 3 hours. The solution was then cooled, slowly poured into cold water (30 ml) and neutralized with 28% ammonia hydride. 100 ml of ethanol was added to the neutral solution, and the obtained filtrate was dried under reduced pressure. The residue was dissolved in water (1.0 ml) and further purified by HPLC under the following conditions. Fractions corresponding to SBThD-F were collected and decompressed to give a white powder (25 mg). Confirmation data of the obtained compound is shown below.
decomp. 265 ° C., NMR (methanol-d 4 ): δ H 8.06 (1H, dd, J = 7.9, J = 4.9), 7.11 (1H, dd, J = 7.9, J = 9.8), ESI-MS: m / z 232.8 [(M−H)].
SBSeD-F and SBThD-F synthesized by the above method are shown in Chemical Formula 4 below.
（１） システイン誘導体の蛍光スペクトル
１ｍＭＥＤＴＡを含む０．１Ｍホウ酸緩衝液（ｐＨ９．０）による上記ＳＢＳｅＤ−Ｆ、ＳＢＴｈＤ−Ｆ又はＳＢＤ−Ｆの各蛍光試薬溶液（４ｍＭ）の５００μｌ部分を、０．１Ｍホウ酸緩衝液（ｐＨ９．０）によるシステイン（０．４ｍＭ）溶液の同量と混合した。この混合物を６０℃で８時間放置した。反応の後、反応混合物をＨＰＬＣ分離し、各システイン誘導体に相当するフラクションを集め、それらの蛍光スペクトルを測定した。
(1) Fluorescence spectrum of
ＳＢＳｅＤ−ＦとＳＢＴｈＤ−Ｆのシステインに対する反応性
４ｍＭの各試薬、ＳＢＳｅＤ−Ｆ、ＳＢＴｈＤ−Ｆ又はＳＢＤ−Ｆ及び１ｍＭ ＥＤＴＡを含む０．１Ｍホウ酸緩衝液（ｐＨ９．０又はｐＨ１０）の５００μｌ部分を、０．１Ｍホウ酸緩衝液（ｐＨ９．０又はｐＨ１０）によるシステイン溶液（０．４ｍＭ）の同量と混合した。反応混合物をＨＰＬＣ分離し、６０℃での反応をモニターした。
Reactivity of SBSeD-F and SBThD-F to cysteine 500 μl portion of 0.1 M borate buffer (pH 9.0 or pH 10) containing 4 mM of each reagent, SBSeD-F, SBThD-F or SBD-F and 1 mM EDTA Was mixed with the same amount of cysteine solution (0.4 mM) in 0.1 M borate buffer (pH 9.0 or pH 10). The reaction mixture was HPLC separated and the reaction at 60 ° C. was monitored.
（２） 結果
ＳＢＤ−Ｆのような水溶性試薬は、そのｓｕｌｆｏｎｉｃ ａｃｉｄ残渣により水溶体中での誘導体の可溶性を増加させ、結果として、誘導体の吸収又は沈殿が減少する。したがって、インシュリンのような比較的疎水性ペプチドのＳＢＤ−Ｆによる誘導体は、逆相カラムで溶出され、高感度に検出されたが、本実施例では、更に、ｂｅｎｚｏｓｅｌｅｎａｄｉａｚｏｌｅ又はｂｅｎｚｏｔｈｉａｄｉａｚｏｌｅ骨格をもつ蛍光試薬としてＳＢＳｅＤ−Ｆ及びＳＢＴｈＤ−Ｆを合成し、それらのシステインに対する反応性及びその誘導体の蛍光特性を調べた。
(2) Results A water-soluble reagent such as SBD-F increases the solubility of the derivative in the aqueous form due to its sulphonic acid residue, resulting in a decrease in absorption or precipitation of the derivative. Accordingly, a derivative of a relatively hydrophobic peptide such as insulin by SBD-F was eluted with a reverse phase column and detected with high sensitivity. In this example, however, as a fluorescent reagent having a benzoselenadiazole or benzothiadiazole skeleton, SBSeD-F and SBThD-F were synthesized, and their reactivity to cysteine and the fluorescence properties of their derivatives were investigated.
最大励起（λｅｘ）及び発光波長（λｅｘ）、及び誘導体のリテンションタイムを表５に示す。各システイン誘導体のｍａｓｓ ｎｕｍｂｅｒ（[Ｍ＋Ｈ]）は、ＳＢＳｅＤ−Ｆ（ｍ／ｚ３８１．９）、ＳＢＴｈＤ−Ｆ（ｍ／ｚ３３４．０）及びＳＢＤ−Ｆ（ｍ／ｚ３１８．０）について、理論値（各々３８２．０、３３４．０及び３１８．０）と一致した。誘導体の最大励起波長は、ＳＢＳｅＤ−Ｆ（３４０ｎｍ）及びＳＢＴｈＤ−Ｆ（３１５ｎｍ）はＳＢＤ−Ｆ（３６５ｎｍ）より短く、誘導体の最大発光波長は、ＳＢＳｅＤ−Ｆ（５４２ｎｍ）は、ＳＢＳｅＤ−Ｆ（５１７ｎｍ）及びＳＢＤ−Ｆ（５１４ｎｍ）よりも長かった。ＳＢＳｅＤ−Ｆ自体は蛍光が少ないが、ＳＢＴｈＤ−Ｆは多少の蛍光を与えた（λｅｘ ；３５０ｎｍ，λｅｍ：４２４ｎｍ）。ＳＢＳｅＤ−Ｆ、ＳＢＴｈＤ−Ｆ及びＳＢＤ−Ｆのシステイン誘導体の逆相カラム（Ｃ１８）に対するｐＨ２．０の移動相によるリテンションタイム（ｔｐ）は、各々４．５、５．３及び４．８分であった。これから、ＳＢＳｅＤ−Ｆは、これらの中で最も親水性の蛍光試薬であった。 Table 5 shows the maximum excitation (λ ex ) and emission wavelength (λ ex ) and the retention time of the derivatives. The mass number ([M + H]) of each cysteine derivative is the theoretical value for SBSeD-F (m / z 381.9), SBThD-F (m / z 334.0) and SBD-F (m / z 318.0). 382.0, 334.0 and 318.0) respectively. The maximum excitation wavelength of the derivative is shorter than SBD-F (365 nm) for SBSeD-F (340 nm) and SBThD-F (315 nm), and the maximum emission wavelength of the derivative is SBSeD-F (517 nm) for SBSeD-F (542 nm). ) And SBD-F (514 nm). SBSeD-F itself has little fluorescence, but SBThD-F gave some fluorescence (λ ex ; 350 nm, λ em : 424 nm). Retention times (tp) with mobile phase at pH 2.0 for reversed phase columns (C 18 ) of cysteine derivatives of SBSeD-F, SBThD-F and SBD-F are 4.5, 5.3 and 4.8 min, respectively. Met. From this, SBSeD-F was the most hydrophilic fluorescent reagent among them.
ＳＢＤ−Ｆの場合、至適反応条件は６０℃でｐＨ９．５で１時間であるが、ＳＢＳｅＤ−Ｆ及びＳＢＴｈＤ−Ｆの反応性は、ＳＢＤ−Ｆと比べて低く、蛍光強度は８−２４時間で徐々に増加し（図９、１０）、２４時間後でも、反応は最大に達しなかった(図９)。ｐＨ１０．０及び６０℃では、ＳＢＳｅＤ−Ｆ又はＳＢＴｈＤ−Ｆとシステインの量的反応時間は、８時間以上であり、ＳＢＤ−Ｆでは、１時間以内で完全に反応した（図１０）。
このように、水溶性の蛍光試薬ＳＢＳｅＤ−Ｆ及びＳＢＴｈＤ−Ｆは、ＳＢＤ−Ｆと比べて蛍光特性及び疎水性の点で異なっており、プロテオーム解析のための新規蛍光誘導体化試薬として有用である。
In the case of SBD-F, the optimum reaction conditions are 60 ° C. and 1 hour at pH 9.5, but the reactivity of SBSeD-F and SBThD-F is lower than that of SBD-F, and the fluorescence intensity is 8-24. It gradually increased with time (FIGS. 9 and 10), and even after 24 hours, the response did not reach a maximum (FIG. 9). At pH 10.0 and 60 ° C., the quantitative reaction time of SBSeD-F or SBThD-F and cysteine was 8 hours or more, and SBD-F reacted completely within 1 hour (FIG. 10).
Thus, the water-soluble fluorescent reagents SBSeD-F and SBThD-F differ from SBD-F in terms of fluorescence characteristics and hydrophobicity, and are useful as novel fluorescent derivatization reagents for proteome analysis. .
ＤＡＡＢＤ−ＣｌによるＣ．ｅｌｅｇａｎｓタンパク質の誘導体化及び同定
（１）方法
Ｃ．ｅｌｅｇａｎｓ（Ｂｒｉｓｔｏｌ Ｎ２株）を、Ｅ．ｃｏｌｉのＯＰ５０株を栄養源として２０℃で、ＮＧＭ寒天上に培養し、Ｍ９バッファーにより浮遊させてバクテリアから分離した。上記線虫をＭ９バッファーで２回洗った後、−８０℃で保管して用いた。この線虫を等量の１０ｍＭ ＣＨＡＰＳに懸濁し、超音波で溶解した。可溶性のフラクションを４℃で１０，０００ｒｐｍ、５分の遠心分離により集めた。上澄を可溶性フラクションとして−２０℃で保管した。このフラクションのタンパク質濃度をＢＳＡを標準に用いるＢｒａｄｆｏｒｄ ｍｅｔｈｏｄで決定した。上澄の約２０μＬ（１００μｇタンパク質）を同容量の２．５ｍＭ ＴＣＥＰ，１７．５ｍＭ ＤＡＡＢＤ−Ｃｌ、１０ｍＭ Ｎａ2 ＥＤＴＡ及び５０ｍＭ ＣＨＡＰＳを６．０Ｍグアニジンを含む１００ｍＭホウ酸塩バッファー（ｐＨ９．０）中で混合した。反応混合物を４０℃で３０分インキュベートした後、反応を２００μＬの０．１％ギ酸で停止し、次いで、反応混合物（１０μｇタンパク質）の３０μＬをＨＰＬＣシステムに注入した。
C. by DAABD-Cl. Derivatization and identification of elegans protein (1) Method elegans (Bristol N2 strain). The OP50 strain of E. coli was cultured on NGM agar at 20 ° C. as a nutrient source and suspended from M9 buffer to separate it from bacteria. The nematode was washed twice with M9 buffer and stored at -80 ° C for use. This nematode was suspended in an equal amount of 10 mM CHAPS and dissolved by ultrasonic waves. Soluble fractions were collected by centrifugation at 10,000 rpm for 5 minutes at 4 ° C. The supernatant was stored at −20 ° C. as a soluble fraction. The protein concentration of this fraction was determined by the Bradford method using BSA as a standard. About 20 μL of the supernatant (100 μg protein) in the same volume of 2.5 mM TCEP, 17.5 mM DAABD-Cl, 10 mM Na 2 EDTA and 50 mM CHAPS in 100 mM borate buffer (pH 9.0) containing 6.0 M guanidine. Mixed. After incubating the reaction mixture at 40 ° C. for 30 minutes, the reaction was stopped with 200 μL of 0.1% formic acid and then 30 μL of the reaction mixture (10 μg protein) was injected into the HPLC system.
ＲＰカラムがＰＲＯＴＥＩＮ（３０ｎｍポアサイズ、２５０×４．６ｍｍ ｉ．ｄ．）（Ｉｍｔａｋｔ）、移動相が溶離液（Ａ）０．１％トリフルオロ酢酸及び溶離液（Ｂ）水／ＣＨ3 ＣＮ／トリフルオロ酢酸（７０／３０／０．１）、グラジエントシステムが流速０．２５ｍＬ／ｍｉｎで１００分以上の３０から７０％Ｂの条件でＨＰＬＣを行った。蛍光検出は５０８nm、励起波長は３８７nmで行った。同定のために、蛍光タンパク質誘導体のいくつかのピークフラクションを分離し、減圧下に１０μＬに濃縮した。各フラクションは、２μｇ／ｍＬトリプシン及び１．０ｍＭ塩化カルシウムを含む９０μＬの５．０ｍＭ重炭酸アンモニウム溶液（ｐＨ７．８）で希釈し、３７℃で２時間インキュベートした。各タンパク質加水分解ペプチド混合物を直接ＥＳＩイオントラップ質量分析装置を用いたＬＣ−ＭＳ／ＭＳに供した。クロマトグラフィーは、ＨＰ１０９０シリーズIIシステム及びＣａｄｅｎｚａ ＴＣ−１８ ｃｏｌｕｍｎ（１２ｎｍポーラスシリカ、１００×２．０ｍｍ ｉ．ｄ．）のカラムを用いて実施した。移動相は溶離液（Ａ）１．０ｍＭギ酸アンモニウム及び溶離液（Ｂ）１．０ｍＭギ酸アンモニウム／ＣＨ3 ＣＮ（５０／５０）とした。グラジエント溶出は、流速０．２ｍＬ／ｍｉｎで６０分以上の０から１００％で行った。タンパク質の同定は、システインのチオール残基に結合したＤＡＡＢＤを記憶するＭＡＳＣＯＴ（Ｍａｔｒｉｘ Ｓｃｉｅｎｃｅ Ｌｔｄ．，Ｕ．Ｋ．）データベースサーチアルゴリズムを有するＮＣＢＩｎｒデータベースを用いて実施した。 RP column is PROTEIN (30 nm pore size, 250 × 4.6 mm id) (Imtakt), mobile phase is eluent (A) 0.1% trifluoroacetic acid and eluent (B) water / CH 3 CN / tri Fluoroacetic acid (70/30 / 0.1), HPLC was performed under the conditions of 30 to 70% B over 100 minutes at a flow rate of 0.25 mL / min with a gradient system. The fluorescence detection was performed at 508 nm and the excitation wavelength was 387 nm. For identification, several peak fractions of the fluorescent protein derivative were separated and concentrated to 10 μL under reduced pressure. Each fraction was diluted with 90 μL of 5.0 mM ammonium bicarbonate solution (pH 7.8) containing 2 μg / mL trypsin and 1.0 mM calcium chloride and incubated at 37 ° C. for 2 hours. Each protein hydrolyzed peptide mixture was directly subjected to LC-MS / MS using an ESI ion trap mass spectrometer. Chromatography was performed using a column of HP1090 series II system and Cadenza TC-18 column (12 nm porous silica, 100 × 2.0 mm id). The mobile phases were eluent (A) 1.0 mM ammonium formate and eluent (B) 1.0 mM ammonium formate / CH 3 CN (50/50). Gradient elution was performed from 0 to 100% over 60 minutes at a flow rate of 0.2 mL / min. Protein identification was performed using the NCBInr database with the MASCOT (Matrix Science Ltd., UK) database search algorithm that stores DAABD bound to the thiol residue of cysteine.
（２）結果
図１１は、ＤＡＡＢＤ−Ｃｌで誘導体化した、Ｃ．ｅｌｅｇａｎｓの可溶性フラクションから得られたタンパク質（約１０μｇ）のクロマトグラムを示す。本実施例では、タンパク質の分離、トリプシンによる加水分解及び任意に選択されたピークフラクションのＬＣ−ＭＳ／ＭＳ同定により、１０種類のタンパク質が同定された。
図中、１はリボゾームタンパク質Ｓ３ａ（ＭＷ＝２８９４２）、２はカルレティキュリン（ｃａｌｒｅｔｉｃｕｌｉｎ）前駆体（ＭＷ＝４５５８８）、３はリボゾームタンパク質Ｌ１（ＭＷ＝３８６３５）、４は伸長因子（ｅｌｏｎｇａｔｉｏｎ ｆａｃｔｏｒ）１−アルファ（ＭＷ＝５０６３６）、５はリンゴ酸デヒドロゲナーゼ（ＭＷ＝３５０９８）、６は４０Ｓリボゾームタンパク質（ＭＷ＝２２０４４）、７はビテロゲニン（ｖｉｔｅｌｌｏｇｅｎｉｎ）（ＭＷ＝１９３０９８）、８はアルギニンキナーゼ（ＭＷ＝４１９６９）、９はＨＳＰ−１熱ショック（ｈｅａｔ ｓｈｏｃｋ）７０ｋｄタンパク質Ａ（ＭＷ＝６９６８０）及び１０はリボゾームタンパク質Ｌ７Ａｅ（ＭＷ＝１３９９２）を示す。本実施例では、任意に選択された１０種類のタンパク質が同定されたが、本発明では、同様にして、他のタンパク質の同定をすることが可能である。
(2) Results FIG. 11 shows C.I. derivatized with DAABD-Cl. The chromatogram of the protein (about 10 microgram) obtained from the soluble fraction of elegans is shown. In this example, 10 proteins were identified by protein separation, hydrolysis with trypsin, and LC-MS / MS identification of arbitrarily selected peak fractions.
In the figure, 1 is a ribosomal protein S3a (MW = 28942), 2 is a calreticulin precursor (MW = 45588), 3 is a ribosomal protein L1 (MW = 38635), and 4 is an elongation factor. 1-alpha (MW = 50636), 5 is malate dehydrogenase (MW = 35098), 6 is 40S ribosomal protein (MW = 22044), 7 is vitellogenin (MW = 193098), 8 is arginine kinase (MW = 41969), 9 indicates HSP-1 heat shock 70 kd protein A (MW = 69680) and 10 indicates ribosomal protein L7Ae (MW = 13992). In this example, 10 kinds of proteins selected arbitrarily were identified, but in the present invention, other proteins can be identified in the same manner.
以上詳述したように、本発明は、微量の発現タンパク質及び／又はペプチドの検出・分離・同定方法及びそのシステムに係るものであり、本発明により、遺伝子を介して発現する発現タンパク質及び／又はペプチドを簡便な方法及び手段で、高感度に検出・分離・同定することができる。本発明の方法により、従来法では検出できなかった微量の発現タンパク質及び／又はペプチドを短時間で、感度良く検出・分離・同定することができる。また、上記検出・分離・同定方法に使用する微量の発現タンパク質及び／又はペプチドの微量検出・分離・同定システムを提供することができる。本発明は、プロテオームのプラットフォーム技術を提供するものとして有用である。 As described above in detail, the present invention relates to a method for detecting, separating and identifying a small amount of expressed protein and / or peptide, and a system thereof. According to the present invention, the expressed protein and / or expressed via a gene Peptides can be detected, separated and identified with high sensitivity by simple methods and means. According to the method of the present invention, a small amount of expressed protein and / or peptide that could not be detected by the conventional method can be detected, separated and identified with high sensitivity in a short time. In addition, it is possible to provide a minute amount detection / separation / identification system for expressed proteins and / or peptides used in the detection / separation / identification method. The present invention is useful for providing proteome platform technology.
Claims (10)
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2003342681A JP4558297B2 (en) | 2002-10-01 | 2003-09-30 | Detection / separation / identification method of expressed trace protein / peptide |
Applications Claiming Priority (3)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| JP2002289313 | 2002-10-01 | ||
| JP2003160201 | 2003-06-05 | ||
| JP2003342681A JP4558297B2 (en) | 2002-10-01 | 2003-09-30 | Detection / separation / identification method of expressed trace protein / peptide |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| JP2005017264A JP2005017264A (en) | 2005-01-20 |
| JP4558297B2 true JP4558297B2 (en) | 2010-10-06 |
Family
ID=34198690
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| JP2003342681A Expired - Lifetime JP4558297B2 (en) | 2002-10-01 | 2003-09-30 | Detection / separation / identification method of expressed trace protein / peptide |
Country Status (1)
| Country | Link |
|---|---|
| JP (1) | JP4558297B2 (en) |
Families Citing this family (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2007309707A (en) * | 2006-05-16 | 2007-11-29 | Kazuhiro Imai | Fluorescence reagent |
| JP2009236908A (en) * | 2008-03-04 | 2009-10-15 | Kazuhiro Imai | Diagnostic and therapeutic method for breast cancer |
| JP2010071986A (en) * | 2008-08-20 | 2010-04-02 | Kazuhiro Imai | Fluorescence reagent |
| CN105092754B (en) * | 2014-05-21 | 2018-09-18 | 天津市汉康医药生物技术有限公司 | A method of measuring sulfonic acid esters genotoxicity impurity using HPLC |
| WO2023140228A1 (en) * | 2022-01-18 | 2023-07-27 | 国立大学法人東北大学 | Method for visualizing modified state or aggregated state of protein |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001235477A (en) * | 2000-02-23 | 2001-08-31 | Natl Inst Of Advanced Industrial Science & Technology Meti | Method for analyzing sequence of peptide amino acid |
-
2003
- 2003-09-30 JP JP2003342681A patent/JP4558297B2/en not_active Expired - Lifetime
Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| JP2001235477A (en) * | 2000-02-23 | 2001-08-31 | Natl Inst Of Advanced Industrial Science & Technology Meti | Method for analyzing sequence of peptide amino acid |
Also Published As
| Publication number | Publication date |
|---|---|
| JP2005017264A (en) | 2005-01-20 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| JP4300029B2 (en) | Method and apparatus for gel-free qualitative and quantitative proteomic analysis and use thereof | |
| US7635573B2 (en) | Mass spectroscopic method for comparing protein levels in two or more samples | |
| US20180052174A1 (en) | Methods and reagents for biomolecule labeling, enrichment and gentle elution | |
| US20110028330A1 (en) | Compounds and methods for the labelling and affinity-selection of proteins | |
| JP4679368B2 (en) | Detection / separation / identification method of expressed trace protein / peptide | |
| JP4558297B2 (en) | Detection / separation / identification method of expressed trace protein / peptide | |
| US20100167262A1 (en) | Method and reagent for the specific identification and quantification of one or more proteins in a sample using in particular inductively coupled plasma-mass spectrometry | |
| US7244411B2 (en) | Method of selective peptide isolation for the identification and quantitative analysis of proteins in complex mixtures | |
| EP2545384B1 (en) | Method for determining the concentration of a peptide | |
| JP4271687B2 (en) | Labeling substance for mass spectrometry and phosphorylation position analysis of phosphorylated protein | |
| US20050042676A1 (en) | Characterising polypeptides | |
| WO2006073563A2 (en) | Fluorescent affinity tag to enhance phosphoprotein detection | |
| US20070128729A1 (en) | Method for the identification and relative quantification of proteins based on the selective isolation of RRnK peptides for the simplification of complex mixtures of proteins | |
| EP4130022A1 (en) | Phosphorylated protein labeling reagent containing equal-weight stable isotopes, preparation method therefor and use thereof | |
| Guo et al. | A novel quantitative proteomics reagent based on soluble nanopolymers | |
| WO2004013636A2 (en) | Differential labeling for quantitative analysis of complex protein mixtures by trifunctional synthetic peptide based reagents with iodoacetamido groups | |
| WO2005015239A2 (en) | Method for n-terminal labeling of proteins | |
| CN116337567A (en) | Quick and efficient chemical proteomics sample preparation method | |
| CN117946081A (en) | Mass spectrum cleavable abnormal-shaped difunctional crosslinking agent and preparation method and application thereof | |
| KR20050079558A (en) | Selective labeling agent for phosphoproteome analysis and phosphorylated site analysis | |
| Stone et al. | Identification of proteins based on MS/MS spectra and location of posttranslational modifications | |
| JP2005291836A (en) | Functional tag for concentrating target substance and method for using the same | |
| AU2002310610A1 (en) | Characterising polypeptides |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20061002 |
|
| A621 | Written request for application examination |
Free format text: JAPANESE INTERMEDIATE CODE: A621 Effective date: 20061002 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20091007 |
|
| A601 | Written request for extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A601 Effective date: 20091207 |
|
| A602 | Written permission of extension of time |
Free format text: JAPANESE INTERMEDIATE CODE: A602 Effective date: 20091210 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100106 |
|
| A131 | Notification of reasons for refusal |
Free format text: JAPANESE INTERMEDIATE CODE: A131 Effective date: 20100421 |
|
| A521 | Request for written amendment filed |
Free format text: JAPANESE INTERMEDIATE CODE: A523 Effective date: 20100616 |
|
| TRDD | Decision of grant or rejection written | ||
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 Effective date: 20100708 |
|
| A01 | Written decision to grant a patent or to grant a registration (utility model) |
Free format text: JAPANESE INTERMEDIATE CODE: A01 |
|
| A61 | First payment of annual fees (during grant procedure) |
Free format text: JAPANESE INTERMEDIATE CODE: A61 Effective date: 20100721 |
|
| R150 | Certificate of patent or registration of utility model |
Ref document number: 4558297 Country of ref document: JP Free format text: JAPANESE INTERMEDIATE CODE: R150 Free format text: JAPANESE INTERMEDIATE CODE: R150 |
|
| FPAY | Renewal fee payment (event date is renewal date of database) |
Free format text: PAYMENT UNTIL: 20130730 Year of fee payment: 3 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| R250 | Receipt of annual fees |
Free format text: JAPANESE INTERMEDIATE CODE: R250 |
|
| EXPY | Cancellation because of completion of term |