HRP20080563A2 - Proteomics profiles and detection methods of proteins in blood plasma - Google Patents
Proteomics profiles and detection methods of proteins in blood plasma Download PDFInfo
- Publication number
- HRP20080563A2 HRP20080563A2 HR20080563A HRP20080563A HRP20080563A2 HR P20080563 A2 HRP20080563 A2 HR P20080563A2 HR 20080563 A HR20080563 A HR 20080563A HR P20080563 A HRP20080563 A HR P20080563A HR P20080563 A2 HRP20080563 A2 HR P20080563A2
- Authority
- HR
- Croatia
- Prior art keywords
- expression
- sample
- precursor
- control
- proteomic profile
- Prior art date
Links
- 108090000623 proteins and genes Proteins 0.000 title claims abstract description 75
- 102000004169 proteins and genes Human genes 0.000 title claims abstract description 75
- 210000002381 plasma Anatomy 0.000 title claims description 27
- 238000001514 detection method Methods 0.000 title description 2
- 102000005234 Adenosylhomocysteinase Human genes 0.000 claims abstract description 63
- 108020002202 Adenosylhomocysteinase Proteins 0.000 claims abstract description 63
- 238000000034 method Methods 0.000 claims abstract description 58
- 230000007812 deficiency Effects 0.000 claims abstract description 32
- 230000014509 gene expression Effects 0.000 claims description 67
- 239000002243 precursor Substances 0.000 claims description 57
- 239000012634 fragment Substances 0.000 claims description 27
- 208000027219 Deficiency disease Diseases 0.000 claims description 22
- 102000050760 Vitamin D-binding protein Human genes 0.000 claims description 17
- 101710179590 Vitamin D-binding protein Proteins 0.000 claims description 17
- 238000000539 two dimensional gel electrophoresis Methods 0.000 claims description 16
- 108010059886 Apolipoprotein A-I Proteins 0.000 claims description 9
- 102000005666 Apolipoprotein A-I Human genes 0.000 claims description 9
- 102100037320 Apolipoprotein A-IV Human genes 0.000 claims description 9
- 102100024520 Ficolin-3 Human genes 0.000 claims description 9
- 101710155250 Ficolin-3 Proteins 0.000 claims description 9
- 101000998953 Homo sapiens Immunoglobulin heavy variable 1-2 Proteins 0.000 claims description 9
- 101001135088 Homo sapiens LIM domain only protein 7 Proteins 0.000 claims description 9
- 102100036887 Immunoglobulin heavy variable 1-2 Human genes 0.000 claims description 9
- 102100033515 LIM domain only protein 7 Human genes 0.000 claims description 9
- 108010071690 Prealbumin Proteins 0.000 claims description 9
- 102100036782 Serine/threonine-protein phosphatase 2A activator Human genes 0.000 claims description 9
- 101710196539 Serine/threonine-protein phosphatase 2A activator Proteins 0.000 claims description 9
- 102000004338 Transferrin Human genes 0.000 claims description 9
- 108090000901 Transferrin Proteins 0.000 claims description 9
- 102000009190 Transthyretin Human genes 0.000 claims description 9
- 108010073614 apolipoprotein A-IV Proteins 0.000 claims description 9
- 102000011778 gamma-delta T-Cell Antigen Receptors Human genes 0.000 claims description 9
- 108010062214 gamma-delta T-Cell Antigen Receptors Proteins 0.000 claims description 9
- 102000014702 Haptoglobin Human genes 0.000 claims description 6
- 108050005077 Haptoglobin Proteins 0.000 claims description 6
- 102100021144 Zinc-alpha-2-glycoprotein Human genes 0.000 claims description 6
- 101710201241 Zinc-alpha-2-glycoprotein Proteins 0.000 claims description 6
- 238000004949 mass spectrometry Methods 0.000 claims description 5
- 238000001502 gel electrophoresis Methods 0.000 claims description 4
- 229930003316 Vitamin D Natural products 0.000 claims 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 claims 1
- 235000019166 vitamin D Nutrition 0.000 claims 1
- 239000011710 vitamin D Substances 0.000 claims 1
- 150000003710 vitamin D derivatives Chemical class 0.000 claims 1
- 229940046008 vitamin d Drugs 0.000 claims 1
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 abstract description 20
- 201000010099 disease Diseases 0.000 abstract description 14
- 238000011161 development Methods 0.000 abstract description 4
- 230000018109 developmental process Effects 0.000 abstract description 4
- 239000012472 biological sample Substances 0.000 abstract description 2
- 230000008859 change Effects 0.000 abstract description 2
- 230000000694 effects Effects 0.000 abstract description 2
- 229920002521 macromolecule Polymers 0.000 abstract description 2
- 230000035790 physiological processes and functions Effects 0.000 abstract description 2
- 239000000523 sample Substances 0.000 description 58
- 235000018102 proteins Nutrition 0.000 description 53
- 239000000499 gel Substances 0.000 description 27
- 238000004458 analytical method Methods 0.000 description 19
- 238000001962 electrophoresis Methods 0.000 description 12
- XSQUKJJJFZCRTK-UHFFFAOYSA-N Urea Chemical compound NC(N)=O XSQUKJJJFZCRTK-UHFFFAOYSA-N 0.000 description 10
- 239000000872 buffer Substances 0.000 description 10
- 238000012360 testing method Methods 0.000 description 10
- ZJUKTBDSGOFHSH-WFMPWKQPSA-N S-Adenosylhomocysteine Chemical compound O[C@@H]1[C@H](O)[C@@H](CSCC[C@H](N)C(O)=O)O[C@H]1N1C2=NC=NC(N)=C2N=C1 ZJUKTBDSGOFHSH-WFMPWKQPSA-N 0.000 description 9
- 230000035772 mutation Effects 0.000 description 9
- 238000013459 approach Methods 0.000 description 6
- 238000004445 quantitative analysis Methods 0.000 description 6
- 238000000926 separation method Methods 0.000 description 6
- 238000002560 therapeutic procedure Methods 0.000 description 6
- 102000004506 Blood Proteins Human genes 0.000 description 5
- 108010017384 Blood Proteins Proteins 0.000 description 5
- MEFKEPWMEQBLKI-AIRLBKTGSA-N S-adenosyl-L-methioninate Chemical compound O[C@@H]1[C@H](O)[C@@H](C[S+](CC[C@H](N)C([O-])=O)C)O[C@H]1N1C2=NC=NC(N)=C2N=C1 MEFKEPWMEQBLKI-AIRLBKTGSA-N 0.000 description 5
- 229960001570 ademetionine Drugs 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 5
- 238000002405 diagnostic procedure Methods 0.000 description 5
- 208000035475 disorder Diseases 0.000 description 5
- VHJLVAABSRFDPM-QWWZWVQMSA-N dithiothreitol Chemical compound SC[C@@H](O)[C@H](O)CS VHJLVAABSRFDPM-QWWZWVQMSA-N 0.000 description 5
- 238000001155 isoelectric focusing Methods 0.000 description 5
- 238000007069 methylation reaction Methods 0.000 description 5
- 239000000203 mixture Substances 0.000 description 5
- 238000004451 qualitative analysis Methods 0.000 description 5
- 229920000936 Agarose Polymers 0.000 description 4
- DHMQDGOQFOQNFH-UHFFFAOYSA-N Glycine Chemical compound NCC(O)=O DHMQDGOQFOQNFH-UHFFFAOYSA-N 0.000 description 4
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 4
- 210000004369 blood Anatomy 0.000 description 4
- 239000008280 blood Substances 0.000 description 4
- UDSAIICHUKSCKT-UHFFFAOYSA-N bromophenol blue Chemical compound C1=C(Br)C(O)=C(Br)C=C1C1(C=2C=C(Br)C(O)=C(Br)C=2)C2=CC=CC=C2S(=O)(=O)O1 UDSAIICHUKSCKT-UHFFFAOYSA-N 0.000 description 4
- 239000004202 carbamide Substances 0.000 description 4
- 238000003384 imaging method Methods 0.000 description 4
- 230000011987 methylation Effects 0.000 description 4
- 238000012544 monitoring process Methods 0.000 description 4
- 239000000243 solution Substances 0.000 description 4
- UMGDCJDMYOKAJW-UHFFFAOYSA-N thiourea Chemical compound NC(N)=S UMGDCJDMYOKAJW-UHFFFAOYSA-N 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
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 3
- 101150057747 SAHH gene Proteins 0.000 description 3
- 239000007983 Tris buffer Substances 0.000 description 3
- 235000001014 amino acid Nutrition 0.000 description 3
- 150000001413 amino acids Chemical class 0.000 description 3
- 239000003153 chemical reaction reagent Substances 0.000 description 3
- 238000001816 cooling Methods 0.000 description 3
- 239000006167 equilibration buffer Substances 0.000 description 3
- PGLTVOMIXTUURA-UHFFFAOYSA-N iodoacetamide Chemical compound NC(=O)CI PGLTVOMIXTUURA-UHFFFAOYSA-N 0.000 description 3
- 238000001840 matrix-assisted laser desorption--ionisation time-of-flight mass spectrometry Methods 0.000 description 3
- 239000013610 patient sample Substances 0.000 description 3
- 239000010979 ruby Substances 0.000 description 3
- 229910001750 ruby Inorganic materials 0.000 description 3
- 238000010561 standard procedure Methods 0.000 description 3
- LENZDBCJOHFCAS-UHFFFAOYSA-N tris Chemical compound OCC(N)(CO)CO LENZDBCJOHFCAS-UHFFFAOYSA-N 0.000 description 3
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Chemical compound O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 3
- QTBSBXVTEAMEQO-UHFFFAOYSA-N Acetic acid Chemical compound CC(O)=O QTBSBXVTEAMEQO-UHFFFAOYSA-N 0.000 description 2
- HRPVXLWXLXDGHG-UHFFFAOYSA-N Acrylamide Chemical compound NC(=O)C=C HRPVXLWXLXDGHG-UHFFFAOYSA-N 0.000 description 2
- 108010088751 Albumins Proteins 0.000 description 2
- 102000009027 Albumins Human genes 0.000 description 2
- 108020004705 Codon Proteins 0.000 description 2
- 108020004414 DNA Proteins 0.000 description 2
- 239000004471 Glycine Substances 0.000 description 2
- FFEARJCKVFRZRR-BYPYZUCNSA-N L-methionine Chemical compound CSCC[C@H](N)C(O)=O FFEARJCKVFRZRR-BYPYZUCNSA-N 0.000 description 2
- OUYCCCASQSFEME-QMMMGPOBSA-N L-tyrosine Chemical compound OC(=O)[C@@H](N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-QMMMGPOBSA-N 0.000 description 2
- KWYHDKDOAIKMQN-UHFFFAOYSA-N N,N,N',N'-tetramethylethylenediamine Chemical compound CN(C)CCN(C)C KWYHDKDOAIKMQN-UHFFFAOYSA-N 0.000 description 2
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 2
- ROOXNKNUYICQNP-UHFFFAOYSA-N ammonium persulfate Chemical compound [NH4+].[NH4+].[O-]S(=O)(=O)OOS([O-])(=O)=O ROOXNKNUYICQNP-UHFFFAOYSA-N 0.000 description 2
- 229910001870 ammonium persulfate Inorganic materials 0.000 description 2
- 239000000959 ampholyte mixture Substances 0.000 description 2
- 238000009534 blood test Methods 0.000 description 2
- 239000007853 buffer solution Substances 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- CVSVTCORWBXHQV-UHFFFAOYSA-N creatine Chemical compound NC(=[NH2+])N(C)CC([O-])=O CVSVTCORWBXHQV-UHFFFAOYSA-N 0.000 description 2
- 235000018417 cysteine Nutrition 0.000 description 2
- XUJNEKJLAYXESH-UHFFFAOYSA-N cysteine Natural products SCC(N)C(O)=O XUJNEKJLAYXESH-UHFFFAOYSA-N 0.000 description 2
- 238000003745 diagnosis Methods 0.000 description 2
- 230000037213 diet Effects 0.000 description 2
- 235000005911 diet Nutrition 0.000 description 2
- 239000012153 distilled water Substances 0.000 description 2
- 239000007850 fluorescent dye Substances 0.000 description 2
- 238000012252 genetic analysis Methods 0.000 description 2
- 239000011521 glass Substances 0.000 description 2
- 235000011187 glycerol Nutrition 0.000 description 2
- 208000006454 hepatitis Diseases 0.000 description 2
- 231100000283 hepatitis Toxicity 0.000 description 2
- 238000004811 liquid chromatography Methods 0.000 description 2
- 239000006166 lysate Substances 0.000 description 2
- 208000030159 metabolic disease Diseases 0.000 description 2
- 239000002207 metabolite Substances 0.000 description 2
- 229930182817 methionine Natural products 0.000 description 2
- 239000002480 mineral oil Substances 0.000 description 2
- 235000010446 mineral oil Nutrition 0.000 description 2
- ZIUHHBKFKCYYJD-UHFFFAOYSA-N n,n'-methylenebisacrylamide Chemical compound C=CC(=O)NCNC(=O)C=C ZIUHHBKFKCYYJD-UHFFFAOYSA-N 0.000 description 2
- WTJKGGKOPKCXLL-RRHRGVEJSA-N phosphatidylcholine Chemical compound CCCCCCCCCCCCCCCC(=O)OC[C@H](COP([O-])(=O)OCC[N+](C)(C)C)OC(=O)CCCCCCCC=CCCCCCCCC WTJKGGKOPKCXLL-RRHRGVEJSA-N 0.000 description 2
- 229920002401 polyacrylamide Polymers 0.000 description 2
- 238000003752 polymerase chain reaction Methods 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 238000002731 protein assay Methods 0.000 description 2
- 238000000575 proteomic method Methods 0.000 description 2
- 230000008132 psychomotor development Effects 0.000 description 2
- 238000011002 quantification Methods 0.000 description 2
- 239000000126 substance Substances 0.000 description 2
- OUYCCCASQSFEME-UHFFFAOYSA-N tyrosine Natural products OC(=O)C(N)CC1=CC=C(O)C=C1 OUYCCCASQSFEME-UHFFFAOYSA-N 0.000 description 2
- 108091032973 (ribonucleotides)n+m Proteins 0.000 description 1
- DGZSVBBLLGZHSF-UHFFFAOYSA-N 4,4-diethylpiperidine Chemical compound CCC1(CC)CCNCC1 DGZSVBBLLGZHSF-UHFFFAOYSA-N 0.000 description 1
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 1
- ZHGNHOOVYPHPNJ-UHFFFAOYSA-N Amigdalin Chemical compound FC(F)(F)C(=O)OCC1OC(OCC2OC(OC(C#N)C3=CC=CC=C3)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C2OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C(OC(=O)C(F)(F)F)C1OC(=O)C(F)(F)F ZHGNHOOVYPHPNJ-UHFFFAOYSA-N 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-N Betaine Natural products C[N+](C)(C)CC([O-])=O KWIUHFFTVRNATP-UHFFFAOYSA-N 0.000 description 1
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 1
- 208000032170 Congenital Abnormalities Diseases 0.000 description 1
- 102000004420 Creatine Kinase Human genes 0.000 description 1
- 108010042126 Creatine kinase Proteins 0.000 description 1
- NZNMSOFKMUBTKW-UHFFFAOYSA-N Cyclohexanecarboxylic acid Natural products OC(=O)C1CCCCC1 NZNMSOFKMUBTKW-UHFFFAOYSA-N 0.000 description 1
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 1
- BDAGIHXWWSANSR-UHFFFAOYSA-N Formic acid Chemical compound OC=O BDAGIHXWWSANSR-UHFFFAOYSA-N 0.000 description 1
- 108010033040 Histones Proteins 0.000 description 1
- 108700022128 Hypermethioninemia Proteins 0.000 description 1
- 206010021118 Hypotonia Diseases 0.000 description 1
- QIVBCDIJIAJPQS-VIFPVBQESA-N L-tryptophane Chemical compound C1=CC=C2C(C[C@H](N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-VIFPVBQESA-N 0.000 description 1
- 208000036626 Mental retardation Diseases 0.000 description 1
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 1
- 208000007379 Muscle Hypotonia Diseases 0.000 description 1
- 208000021642 Muscular disease Diseases 0.000 description 1
- 102000006386 Myelin Proteins Human genes 0.000 description 1
- 108010083674 Myelin Proteins Proteins 0.000 description 1
- 201000009623 Myopathy Diseases 0.000 description 1
- KWIUHFFTVRNATP-UHFFFAOYSA-O N,N,N-trimethylglycinium Chemical compound C[N+](C)(C)CC(O)=O KWIUHFFTVRNATP-UHFFFAOYSA-O 0.000 description 1
- FFDGPVCHZBVARC-UHFFFAOYSA-N N,N-dimethylglycine Chemical compound CN(C)CC(O)=O FFDGPVCHZBVARC-UHFFFAOYSA-N 0.000 description 1
- 101710163270 Nuclease Proteins 0.000 description 1
- NBIIXXVUZAFLBC-UHFFFAOYSA-N Phosphoric acid Chemical compound OP(O)(O)=O NBIIXXVUZAFLBC-UHFFFAOYSA-N 0.000 description 1
- 102000001708 Protein Isoforms Human genes 0.000 description 1
- 108010029485 Protein Isoforms Proteins 0.000 description 1
- 102000055027 Protein Methyltransferases Human genes 0.000 description 1
- 102100027378 Prothrombin Human genes 0.000 description 1
- 108010094028 Prothrombin Proteins 0.000 description 1
- 102000004389 Ribonucleoproteins Human genes 0.000 description 1
- 108010081734 Ribonucleoproteins Proteins 0.000 description 1
- 208000031929 S-adenosylhomocysteine hydrolase deficiency Diseases 0.000 description 1
- DBMJMQXJHONAFJ-UHFFFAOYSA-M Sodium laurylsulphate Chemical compound [Na+].CCCCCCCCCCCCOS([O-])(=O)=O DBMJMQXJHONAFJ-UHFFFAOYSA-M 0.000 description 1
- 101000983177 Streptomyces rimosus N,N-dimethyltransferase OxyT Proteins 0.000 description 1
- 102000003929 Transaminases Human genes 0.000 description 1
- 108090000340 Transaminases Proteins 0.000 description 1
- 229920004890 Triton X-100 Polymers 0.000 description 1
- 239000013504 Triton X-100 Substances 0.000 description 1
- 102000004142 Trypsin Human genes 0.000 description 1
- 108090000631 Trypsin Proteins 0.000 description 1
- QIVBCDIJIAJPQS-UHFFFAOYSA-N Tryptophan Natural products C1=CC=C2C(CC(N)C(O)=O)=CNC2=C1 QIVBCDIJIAJPQS-UHFFFAOYSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- 238000009825 accumulation Methods 0.000 description 1
- 230000009471 action Effects 0.000 description 1
- 239000012670 alkaline solution Substances 0.000 description 1
- AFVLVVWMAFSXCK-VMPITWQZSA-N alpha-cyano-4-hydroxycinnamic acid Chemical compound OC(=O)C(\C#N)=C\C1=CC=C(O)C=C1 AFVLVVWMAFSXCK-VMPITWQZSA-N 0.000 description 1
- 235000012538 ammonium bicarbonate Nutrition 0.000 description 1
- 235000019395 ammonium persulphate Nutrition 0.000 description 1
- BFNBIHQBYMNNAN-UHFFFAOYSA-N ammonium sulfate Chemical compound N.N.OS(O)(=O)=O BFNBIHQBYMNNAN-UHFFFAOYSA-N 0.000 description 1
- 229910052921 ammonium sulfate Inorganic materials 0.000 description 1
- 235000011130 ammonium sulphate Nutrition 0.000 description 1
- 229960003237 betaine Drugs 0.000 description 1
- 230000033228 biological regulation Effects 0.000 description 1
- 230000015572 biosynthetic process Effects 0.000 description 1
- 229940098773 bovine serum albumin Drugs 0.000 description 1
- 210000004556 brain Anatomy 0.000 description 1
- 238000005251 capillar electrophoresis Methods 0.000 description 1
- 230000006037 cell lysis Effects 0.000 description 1
- 230000009391 cell specific gene expression Effects 0.000 description 1
- 230000030570 cellular localization Effects 0.000 description 1
- 229960001231 choline Drugs 0.000 description 1
- OEYIOHPDSNJKLS-UHFFFAOYSA-N choline Chemical compound C[N+](C)(C)CCO OEYIOHPDSNJKLS-UHFFFAOYSA-N 0.000 description 1
- 150000001875 compounds Chemical class 0.000 description 1
- 238000004590 computer program Methods 0.000 description 1
- 238000012790 confirmation Methods 0.000 description 1
- 239000013068 control sample Substances 0.000 description 1
- RSJOBNMOMQFPKQ-UHFFFAOYSA-L copper;2,3-dihydroxybutanedioate Chemical compound [Cu+2].[O-]C(=O)C(O)C(O)C([O-])=O RSJOBNMOMQFPKQ-UHFFFAOYSA-L 0.000 description 1
- 229960003624 creatine Drugs 0.000 description 1
- 239000006046 creatine Substances 0.000 description 1
- 238000003748 differential diagnosis Methods 0.000 description 1
- 230000029087 digestion Effects 0.000 description 1
- 108700003601 dimethylglycine Proteins 0.000 description 1
- 229940042399 direct acting antivirals protease inhibitors Drugs 0.000 description 1
- 230000008034 disappearance Effects 0.000 description 1
- 238000011067 equilibration Methods 0.000 description 1
- 235000019441 ethanol Nutrition 0.000 description 1
- 230000003203 everyday effect Effects 0.000 description 1
- 235000019253 formic acid Nutrition 0.000 description 1
- 230000002068 genetic effect Effects 0.000 description 1
- 235000013905 glycine and its sodium salt Nutrition 0.000 description 1
- 239000011544 gradient gel Substances 0.000 description 1
- BPMFZUMJYQTVII-UHFFFAOYSA-N guanidinoacetic acid Chemical compound NC(=N)NCC(O)=O BPMFZUMJYQTVII-UHFFFAOYSA-N 0.000 description 1
- 238000010438 heat treatment Methods 0.000 description 1
- 238000004128 high performance liquid chromatography Methods 0.000 description 1
- 230000007062 hydrolysis Effects 0.000 description 1
- 238000006460 hydrolysis reaction Methods 0.000 description 1
- 238000007654 immersion Methods 0.000 description 1
- 239000012535 impurity Substances 0.000 description 1
- 238000000126 in silico method Methods 0.000 description 1
- 239000003112 inhibitor Substances 0.000 description 1
- 238000004255 ion exchange chromatography Methods 0.000 description 1
- 150000002632 lipids Chemical class 0.000 description 1
- 210000004185 liver Anatomy 0.000 description 1
- 239000000463 material Substances 0.000 description 1
- 239000011159 matrix material Substances 0.000 description 1
- 238000000816 matrix-assisted laser desorption--ionisation Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- VNWKTOKETHGBQD-UHFFFAOYSA-N methane Natural products C VNWKTOKETHGBQD-UHFFFAOYSA-N 0.000 description 1
- 125000002496 methyl group Chemical group [H]C([H])([H])* 0.000 description 1
- -1 minimum 99% Chemical compound 0.000 description 1
- 230000004048 modification Effects 0.000 description 1
- 238000012986 modification Methods 0.000 description 1
- 238000001964 muscle biopsy Methods 0.000 description 1
- 210000005012 myelin Anatomy 0.000 description 1
- 230000023105 myelination Effects 0.000 description 1
- 238000010606 normalization Methods 0.000 description 1
- 230000003287 optical effect Effects 0.000 description 1
- 239000002245 particle Substances 0.000 description 1
- 239000000137 peptide hydrolase inhibitor Substances 0.000 description 1
- 235000011007 phosphoric acid Nutrition 0.000 description 1
- 230000004481 post-translational protein modification Effects 0.000 description 1
- 230000001323 posttranslational effect Effects 0.000 description 1
- 239000002244 precipitate Substances 0.000 description 1
- 238000001556 precipitation Methods 0.000 description 1
- 230000002035 prolonged effect Effects 0.000 description 1
- 230000006916 protein interaction Effects 0.000 description 1
- 229940039716 prothrombin Drugs 0.000 description 1
- 238000000746 purification Methods 0.000 description 1
- 150000003212 purines Chemical class 0.000 description 1
- 230000011514 reflex Effects 0.000 description 1
- 238000011160 research Methods 0.000 description 1
- 230000004044 response Effects 0.000 description 1
- 238000012552 review Methods 0.000 description 1
- 239000012266 salt solution Substances 0.000 description 1
- 238000012284 sample analysis method Methods 0.000 description 1
- 210000002966 serum Anatomy 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 1
- 235000017557 sodium bicarbonate Nutrition 0.000 description 1
- 238000002415 sodium dodecyl sulfate polyacrylamide gel electrophoresis Methods 0.000 description 1
- RWVGQQGBQSJDQV-UHFFFAOYSA-M sodium;3-[[4-[(e)-[4-(4-ethoxyanilino)phenyl]-[4-[ethyl-[(3-sulfonatophenyl)methyl]azaniumylidene]-2-methylcyclohexa-2,5-dien-1-ylidene]methyl]-n-ethyl-3-methylanilino]methyl]benzenesulfonate Chemical compound [Na+].C1=CC(OCC)=CC=C1NC1=CC=C(C(=C2C(=CC(C=C2)=[N+](CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=2C(=CC(=CC=2)N(CC)CC=2C=C(C=CC=2)S([O-])(=O)=O)C)C=C1 RWVGQQGBQSJDQV-UHFFFAOYSA-M 0.000 description 1
- 238000010186 staining Methods 0.000 description 1
- 238000007447 staining method Methods 0.000 description 1
- 230000001502 supplementing effect Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 238000003786 synthesis reaction Methods 0.000 description 1
- 238000010998 test method Methods 0.000 description 1
- 239000012588 trypsin Substances 0.000 description 1
- 230000029812 viral genome replication Effects 0.000 description 1
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2800/00—Detection or diagnosis of diseases
- G01N2800/60—Complex ways of combining multiple protein biomarkers for diagnosis
Landscapes
- Life Sciences & Earth Sciences (AREA)
- Health & Medical Sciences (AREA)
- Engineering & Computer Science (AREA)
- Molecular Biology (AREA)
- Hematology (AREA)
- Chemical & Material Sciences (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Immunology (AREA)
- Biomedical Technology (AREA)
- Biophysics (AREA)
- Food Science & Technology (AREA)
- Biotechnology (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Microbiology (AREA)
- Bioinformatics & Computational Biology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Cell Biology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
Izum se odnosi na metodu dijagnosticiranja deficijencije S-adenozilhomocistein hidrolaze (SAHH) Predmetni izum pokušava dati jednostavnu, učinkovitu i brzu metodu dijagnosticiranja deficijencije SAHH uz pomoć metoda proteomike. Proteini su ključne makromolekule koje predstavljaju regulatore molekularnih zbivanja u stanici tako da bilo koja promjena proteina ima snažan učinak na fiziološke procese stanice i u konačnici doprinosi razvoju bolesti. Predmetni izum, prema tome daje metodu utvrđivanja proteomskog profila biološkog uzorka subjekta te utvrđuje u odnosu na kontrolne uzorke.The invention relates to a method of diagnosing S-adenosylhomocysteine hydrolase (SAHH) deficiency. The present invention seeks to provide a simple, effective and rapid method for diagnosing SAHH deficiency using proteomics methods. Proteins are key macromolecules that are regulators of molecular developments in the cell such that any change in the protein has a strong effect on the physiological processes of the cell and ultimately contributes to the development of the disease. The present invention therefore provides a method for determining the proteomic profile of a biological sample of a subject and determines it with respect to control samples.
Description
Područje na koje se izum odnosi The field to which the invention relates
Izum se odnosi na metodu dijagnosticiranja deficijencije S-adenozilhomocistein hidrolaze (SAHH) uz pomoć proteomike. Preciznije, predmetni izum se odnosi na proteomsku metodu diferencijalnog dijagnosticiranja deficijencije SAHH. The invention relates to a method of diagnosing S-adenosylhomocysteine hydrolase (SAHH) deficiency with the help of proteomics. More precisely, the present invention relates to the proteomic method of differential diagnosis of SAHH deficiency.
Tehnički problem Technical problem
Deficijencija S-adenozilhomocistein hidrolaze (SAHH) je prvi puta okarakterizirana 2004. godine na pacijentu iz Hrvatske (I. Barić i sur. 2004). Genskom analizom DNA pacijenata utvrđene su dvije mutacije u genu za SAHH, svaka naslijeđena od jednog roditelja. To su dvije točkaste mutacije u eksonu 4. Mutacija naslijeđena od majke nalazi se na aminokiselini Trp112 gdje nastaje stop kodon (TGG – TGA), dok se mutacija naslijeđena od oca nalazi na položaju 143 gdje tirozin prelazi u cistein (TAC -TGC). Nije međutim još razjašnjeno da li osim navedenih mutacija postoje još neke koje pridonose nastanku bolesti i njezinoj kliničkoj slici. Nakon što je bolest okarakterizirana na prvom pacijentu, otkrivena je i kod njegova dva brata (Barić i sur. 2005). 2006. godine dokazan je i četvrti slučaj SAHH deficijencije kod 26-godišnjeg pacijenta. S-adenosylhomocysteine hydrolase (SAHH) deficiency was first characterized in 2004 in a patient from Croatia (I. Barić et al. 2004). Genetic analysis of the patients' DNA revealed two mutations in the SAHH gene, each inherited from one parent. These are two point mutations in exon 4. The mutation inherited from the mother is located at amino acid Trp112 where a stop codon is formed (TGG – TGA), while the mutation inherited from the father is located at position 143 where tyrosine changes to cysteine (TAC -TGC). However, it has not yet been clarified whether, in addition to the mentioned mutations, there are others that contribute to the onset of the disease and its clinical picture. After the disease was characterized in the first patient, it was also discovered in his two brothers (Barić et al. 2005). In 2006, a fourth case of SAHH deficiency was proven in a 26-year-old patient.
Klinička slika bolesti, odnosno djelovanje mutiranog proteina SAHH, vrlo je složena pošto je SAHH izravno povezan sa sintezom aminokiseline metionin iz koje nastaje S-adenozilmetionin (SAM), koji je pak izvor metilne skupine za više od 100 bioloških metilacijskih reakcija (Clarke i Banfield 2001). Za bolest ne postoji adekvatan lijek ali se terapija može provesti dijetom osiromašenom metioninom kako bi se smanjila akumulacija S-adenozilhomocisteina (SAH-a), te nadoknadom kreatinom i fosfatidilkolinom u prehrani. Tretman je do neke mjere pomogao u poboljšanju kliničke slike bolesti, odnosno došlo je do značajnog smanjenja koncentracije SAH-a i SAM-a u plazmi te nestanka tromosti, ubrzanja psihomotornog razvoja te značajnih promjena u mijelinizaciji mozga kod prva dva dijagnosticirana pacijenta (Barić i sur. 2005). The clinical picture of the disease, i.e. the action of the mutated protein SAHH, is very complex since SAHH is directly related to the synthesis of the amino acid methionine from which S-adenosylmethionine (SAM) is formed, which in turn is the source of the methyl group for more than 100 biological methylation reactions (Clarke and Banfield 2001 ). There is no adequate cure for the disease, but therapy can be carried out with a diet depleted of methionine in order to reduce the accumulation of S-adenosylhomocysteine (SAH), and by supplementing it with creatine and phosphatidylcholine in the diet. The treatment helped to some extent in improving the clinical picture of the disease, i.e. there was a significant decrease in the concentration of SAH and SAM in the plasma and the disappearance of sluggishness, acceleration of psychomotor development and significant changes in the myelination of the brain in the first two diagnosed patients (Barić et al. 2005).
Stanje tehnike State of the art
Prilikom prvog dijagnosticiranja poremećaja SAHH, rađene su krvne pretrage pacijenata koje su pokazale hipotoniju sa povećanim serumskim koncentracijama kreatin kinaze i transaminaza, prolongirano protrombinsko vrijeme, nisku koncentracija albumina, povećane vrijednosti SAM i SAH od 30-150 puta u krvi. Ovakav najjednostavniji način pretrage krvi daje rezultate koji upućuju na moguću deficijenciju SAHH, ali također daje rezultate koji bi mogli upućivati i na neke druge bolesti npr. na cijeli niz drugih mogućih metaboličkih poremećaja nastalih kao posljedica urođenih mana ili trenutnog stanja organizma. Prema tome takva inicijalna pretraga krvi bi se morala kombinirati s nekom drugom metodom dijagnosticiranja kako bi se sa sigurnošću utvrdila bolest deficijencije SAHH. When the SAHH disorder was first diagnosed, blood tests were performed on the patients, which showed hypotonia with increased serum concentrations of creatine kinase and transaminases, prolonged prothrombin time, low albumin concentration, increased SAM and SAH values of 30-150 times in the blood. This simplest method of blood testing gives results that point to a possible SAHH deficiency, but it also gives results that could point to some other diseases, for example, to a whole series of other possible metabolic disorders caused as a result of congenital defects or the current state of the organism. Therefore, such an initial blood test would have to be combined with some other diagnostic method in order to determine the SAHH deficiency disease with certainty.
Prilikom daljnjih ispitivanja korištena je kao metoda dijagnosticiranja biopsija mišića. Metoda nije pokazala dobre rezultate ukoliko se samostalno koristila jer bi rezultati takve metode pored poremećaja SAHH upućivali i na abnormalne mijelinske strukture jetre, što je upućivalo na blagi hepatitis. During further tests, muscle biopsies were used as a diagnostic method. The method did not show good results if it was used independently, because the results of such a method would point to abnormal myelin structures of the liver in addition to the SAHH disorder, which indicated mild hepatitis.
Pretrage krvne plazme, kojima se pokušala dobiti jasna dijagnoza poremećaja SAHH, pokazale su osim povećanih vrijednosti SAH-a i niske koncentracije fosfatidilkolina i kolina, povećane koncentracije gvanidino acetata, betaina, dimetilglicina i cistationa. Blood plasma tests, which were used to obtain a clear diagnosis of SAHH disorder, showed, in addition to increased values of SAH and low concentrations of phosphatidylcholine and choline, increased concentrations of guanidino acetate, betaine, dimethylglycine and cystathion.
Nakon svih dijagnostičkih indicija, tek je genska analiza dala konačnu potvrdu bolesti deficijencije S- adenozilhomocistein hidrolaze. Putem metode PCR (lančane reakcije polimeraze) pronađene su kod osoba na koje se sumnjalo da imaju poremećaj enzima SAHH dvije mutacije u genu za SAHH koje su nasljedne, točkaste mutacije u eksonu 4 TGG u TGA (stop kodon namjesto triptofana 112) i TAC u TGC (tirozin 143 u cistein). After all the diagnostic indications, only genetic analysis gave the final confirmation of S-adenosylhomocysteine hydrolase deficiency disease. Through the PCR (polymerase chain reaction) method, two mutations in the SAHH gene were found in individuals suspected of having a disorder of the SAHH enzyme, which are hereditary, point mutations in exon 4 TGG to TGA (stop codon instead of tryptophan 112) and TAC to TGC (tyrosine 143 to cysteine).
Patent WO2006090428 opisuje HPLC tehniku kojom se dijagnosticira cijeli niz metabolita (purine, pririmidine, N-acetilirane amino kiseline, sulfonirane i nitrolizirane spojeve) u biološkom uzorku. Dokument navodi da bi se ovakvim postupkom mogla dijagnosticirati i deficijencija SAHH ali ne ukazuje kako bi se točno izveo taj postupak niti upućuje, s obzirom na količinu detektiranih metabolita u uzorku, koja bi se još metoda dijagnosticiranja morala kombinirati s opisanom metodom kako bi se sa sigurnošću mogla dijagnosticirati deficijencije SAHH. Patent WO2006090428 describes an HPLC technique that diagnoses a whole series of metabolites (purines, pririmidines, N-acetylated amino acids, sulfonated and nitrosated compounds) in a biological sample. The document states that SAHH deficiency could also be diagnosed with this procedure, but it does not indicate how exactly this procedure would be performed, nor does it indicate, considering the amount of detected metabolites in the sample, which other diagnostic method would have to be combined with the described method in order to be sure could diagnose SAHH deficiencies.
Izlaganje suštine izuma Presentation of the essence of the invention
Predmetni izum pokušava dati jednostavan, učinkovit i brz način dijagnosticiranja bolesti deficijencije SAHH. The subject invention attempts to provide a simple, effective and rapid way of diagnosing SAHH deficiency disease.
Kako je SAH snažan inhibitor mnogih SAM-ovisnih metiltransferaza, porast razine SAH i smanjenje omjera SAM/SAH mogući su uzroci drugih biokemijskih poremećaja vezane uz metilacijske procese. Hidroliza S-adenozilhomocisteina igra ključnu ulogu u regulaciji biološke metilacije, koje su važne modifikacije DNK, RNK i proteina. Posttranslacijska metilacija važna je za interakcije među proteinima, smještaj unutar stanice i dozrijevanje različitih ribonukloproteina, te vjerojatno za provođenje signala i replikaciju virusa. Uz to, metilacija je važan dio histonskog koda koji regulira stanično-specifične programe genske ekspresije. As SAH is a strong inhibitor of many SAM-dependent methyltransferases, an increase in SAH levels and a decrease in the SAM/SAH ratio are possible causes of other biochemical disorders related to methylation processes. The hydrolysis of S-adenosylhomocysteine plays a key role in the regulation of biological methylation, which are important modifications of DNA, RNA and proteins. Post-translational methylation is important for protein interactions, intracellular localization and maturation of various ribonucleoproteins, and possibly for signal transduction and viral replication. In addition, methylation is an important part of the histone code that regulates cell-specific gene expression programs.
Vanjski pokazatelji bolesti deficijencije SAHH kod pacijenta su blagi hepatitis, nedostatak bazičnih refleksa, miopatija, nezainteresiranost za okolinu, usporen psihomotorni razvoj te, najvjerojatnije, mentalna retardacija. S obzirom na to da su pokazatelji bolesti prisutni kod puno većeg broja osoba kojima nije dijagnosticirana deficijencija SAHH, moguće je da bi se ispitivanjem pacijenata kod kojih bi se po simptomima moglo posumnjati na deficijenciju SAHH, navedena bolest i otkrila. External indicators of SAHH deficiency disease in a patient are mild hepatitis, lack of basic reflexes, myopathy, lack of interest in the environment, slow psychomotor development and, most likely, mental retardation. Considering that the indicators of the disease are present in a much larger number of people who have not been diagnosed with SAHH deficiency, it is possible that by examining patients whose symptoms could be suspected of having SAHH deficiency, the mentioned disease could be detected.
Međutim, primjena genetičke metode koja je jedina do sada dala precizan dijagnostički rezultat u otkrivaju mutacije na genu za SAHH, u svakodnevnoj dijagnostici nije niti pristupačna, niti praktična jer je skupa, komplicirana i dugo traje, a točkaste mutacije koje su otkrivene ne moraju biti jedine. However, the application of the genetic method, which is the only one so far that has provided a precise diagnostic result in the detection of a mutation in the SAHH gene, is neither affordable nor practical in everyday diagnostics because it is expensive, complicated and takes a long time, and the point mutations that have been detected may not be the only ones .
Predmetni izum pokušava dati metodu dijagnostike koja bi se temelja na mnogo jednostavnijem i bržem načinu dijagnosticiranja bolesti SAHH. S obzirom da su proteini ključne makromolekule koje predstavljaju regulatore molekularnih zbivanja u stanici te da bilo koja promjena proteina ima snažan učinak na fiziološke procese stanice i u konačnici doprinosi razvoju bolesti, predmetni izum analizira krvnu plazmu subjekta te utvrđuje promjene u proteomskom profilu oboljelih osoba od deficijencije SAHH. The subject invention attempts to provide a diagnostic method based on a much simpler and faster method of diagnosing SAHH disease. Given that proteins are key macromolecules that represent regulators of molecular events in the cell and that any protein change has a strong effect on the physiological processes of the cell and ultimately contributes to the development of the disease, the subject invention analyzes the subject's blood plasma and determines changes in the proteomic profile of persons suffering from SAHH deficiency .
Analizom uzoraka plazme krvi pomoću standardnih proteomskih metoda kvantitativne i kvalitativne analize kemijskih supstanci došlo se do otkrića proteomskog profila koji je specifičan za deficijenciju SAHH kod oboljelih osoba. Naime, utvrđeno je da određeni proteini krvne plazme različito eksprimiraju kod osobe oboljele od deficijencije SAHH u usporedbi s metabolički zdravom osobom. Utvrđivanjem specifičnog proteomskog profila osoba oboljelih od deficijencije SAHH i njegovom usporedbom s kontrolom, dobila se referentna razlika proteomskog profila koju je poželjno upotrebljavati u metodi dijagnosticiranja bolesti deficijencije SAHH kao i praćenje razvoja bolesti te praćenje odgovora pacijenta na eventualnu terapiju, a koja je pružena predmetnim izumom. Analysis of blood plasma samples using standard proteomic methods of quantitative and qualitative analysis of chemical substances led to the discovery of a proteomic profile that is specific for SAHH deficiency in affected individuals. Namely, it was determined that certain blood plasma proteins are expressed differently in a person suffering from SAHH deficiency compared to a metabolically healthy person. By determining the specific proteomic profile of persons suffering from SAHH deficiency and comparing it with the control, a reference difference of the proteomic profile was obtained, which is preferably used in the method of diagnosing SAHH deficiency disease, as well as monitoring the development of the disease and monitoring the patient's response to possible therapy, which is provided by the subject invention .
Prema tome, predmetni izum se odnosi na metodu dijagnosticiranja deficijencije SAHH koja sadrži korak u kojem se predmetni izum standardnim metodama proteomike određuje proteomski profil ispitivanog uzorka (u daljnjem tekstu „uzorka“). Već podaci o proteomskom profilu a posebno njegova usporedba s proteomskim profilom kontrolnog uzorka metabolički zdrave osobe (u daljnjem tekstu „kontrole“) identificira proteomski profil u kojem je ekspresija proteina proteomskog profila povezana s deficijencijom SAHH-a. Na gore navedeni način identificirani proteomski profil može biti upotrijebljen u metodi dijagnosticiranja bolesti i u metodi dijagnosticiranja faza bolesti deficijencije SAHH kroz životni vijek pacijenta ili nakon primijenjene terapije. Therefore, the subject invention relates to a method for diagnosing SAHH deficiency, which contains a step in which the subject invention determines the proteomic profile of the examined sample (hereinafter referred to as the "sample") using standard proteomics methods. Already the data on the proteomic profile and especially its comparison with the proteomic profile of a control sample of a metabolically healthy person (hereinafter referred to as "controls") identifies a proteomic profile in which the protein expression of the proteomic profile is associated with SAHH deficiency. The proteomic profile identified in the above manner can be used in the method of diagnosing the disease and in the method of diagnosing the phases of the SAHH deficiency disease throughout the patient's lifetime or after the applied therapy.
Prema tome, jedan od koraka u dijagnosticiranju deficijencije SAHH je utvrđivanje odsutnosti, prisutnosti te stupnja eksprimiranja jednog ili više proteina odabranih iz skupine proteina koja sadrži: Apolipoprotein A-IV (prekursor), Fikolin-3 (prekursor), Serotransferin (prekursor), Transtiretin (prekursor), Haptoglobin (prekursor), Cink alfa 2 glikoprotein (prekursor), LOMP protein, Vitamin D vezujući protein prekursor, Apolipoprotein A-I (fragment), Imunoglobulin teški lanac - varijabilna regija (fragment), Vitamin D vezujući protein, varijacija (fragment), Fosfotirozil fosfatazni aktivator, T-stanični receptor delta lanac HE/801 u uzorku. Therefore, one of the steps in diagnosing SAHH deficiency is determining the absence, presence and level of expression of one or more proteins selected from the group of proteins that contains: Apolipoprotein A-IV (precursor), Ficolin-3 (precursor), Serotransferrin (precursor), Transthyretin (precursor), Haptoglobin (precursor), Zinc alpha 2 glycoprotein (precursor), LOMP protein, Vitamin D binding protein precursor, Apolipoprotein A-I (fragment), Immunoglobulin heavy chain - variable region (fragment), Vitamin D binding protein, variation (fragment ), Phosphotyrosyl phosphatase activator, T-cell receptor delta chain HE/801 in the sample.
Nakon određivanja proteomskog profila uzorka, slijedi korak uspoređivanja proteomskog profila uzorka s proteomskim profilom kontrole, gdje se utvrđuje razlika između proteomskog profila uzorka i proteomskog profila kontrole koja indicira deficijenciju SAHH u uzorku. After determining the proteomic profile of the sample, there is a step of comparing the proteomic profile of the sample with the proteomic profile of the control, where the difference between the proteomic profile of the sample and the proteomic profile of the control is determined, indicating SAHH deficiency in the sample.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, razlika između proteomskog profila uzorka i proteomskog profila kontrole se očituje u povećanom stupnju ekspresije skupine proteina u uzorku u odnosu na stupanj ekspresije skupine proteina u kontroli, gdje se skupina proteina sastoji od: Apolipoprotein A-IV (prekursor), Fikolin-3 (prekursor), Serotransferin (prekursor), Transtiretin (prekursor), Apolipoprotein A-I (fragment), Fosfotirozil fosfatazni aktivator, T-stanični receptor delta lanac HE/801. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the difference between the proteomic profile of the sample and the proteomic profile of the control is manifested in the increased level of expression of a group of proteins in the sample compared to the level of expression of a group of proteins in the control, where the group of proteins consists of: Apolipoprotein A-IV (precursor), Ficolin-3 (precursor), Serotransferrin (precursor), Transthyretin (precursor), Apolipoprotein A-I (fragment), Phosphotyrosyl phosphatase activator, T-cell receptor delta chain HE/801.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, razlika između proteomskog profila uzorka i proteomskog profila kontrole se očituje u smanjenom stupnju ekspresije skupine proteina u uzorku u odnosu na stupanj ekspresije skupine proteina u kontroli, gdje se skupina proteina sastoji od: LOMP protein, Vitamin D vezujući protein prekursor, Imunoglobulin teški lanac - varijabilna regija (fragment), Vitamin D vezujući protein, varijacija (fragment),. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the difference between the proteomic profile of the sample and the proteomic profile of the control is manifested in a reduced level of expression of a group of proteins in the sample compared to the level of expression of a group of proteins in the control, where the group of proteins consists of: LOMP protein, Vitamin D binding protein precursor, Immunoglobulin heavy chain - variable region (fragment), Vitamin D binding protein, variation (fragment),.
U koraku određivanja proteomskog profila uzorka, protein Haptoglobin (prekursor) nije nađen u uzorku kod postojanja bolesti deficijencije SAHH u uzorku. Navedeni protein je prisutan samo u kontroli. In the step of determining the proteomic profile of the sample, the protein Haptoglobin (precursor) was not found in the sample in the presence of SAHH deficiency disease in the sample. The mentioned protein is present only in the control.
. .
U koraku određivanja proteomskog profila uzorka, protein Cink alfa 2 glikoprotein (prekursor) je nađen u uzorku kod postojanja bolesti deficijencije SAHH u uzorku. Navedeni protein nije prisutan u kontroli. In the step of determining the proteomic profile of the sample, the protein Zinc alpha 2 glycoprotein (precursor) was found in the sample in the presence of SAHH deficiency disease in the sample. The specified protein is not present in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 5 puta većoj ekspresiji proteina Apolipoprotein A-IV (prekursor) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the established difference is manifested in a 5-fold higher expression of the protein Apolipoprotein A-IV (precursor) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 5 puta većoj ekspresiji proteina Fikolin-3 (prekursor) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the established difference is manifested in a 5-fold higher expression of the protein Ficolin-3 (precursor) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 5 puta većoj ekspresiji proteina Serotransferin (prekursor) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 5-fold higher expression of the protein Serotransferrin (precursor) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 5 puta većoj ekspresiji proteina Transtiretin (prekursor) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 5-fold higher expression of the protein Transthyretin (precursor) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta većoj ekspresiji proteina Apolipoprotein A-I (fragment) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 3-fold higher expression of the Apolipoprotein A-I protein (fragment) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta većoj ekspresiji proteina Fosfotirozil fosfatazni aktivator kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 3-fold higher expression of the protein Phosphotyrosyl phosphatase activator in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta većoj ekspresiji proteina T-stanični receptor delta lanac HE/801 kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 3-fold higher expression of the protein T-cell receptor delta chain HE/801 in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta manjoj ekspresiji proteina LOMP proteina kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the established difference is manifested in a 3-fold lower expression of the LOMP protein in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta manjoj ekspresiji proteina Vitamin D vezujućeg proteina prekursor kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the established difference is manifested in a 3-fold lower expression of the protein Vitamin D binding protein precursor in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta manjoj ekspresiji proteina Imunoglobulin teški lanac - varijabilna regija (fragment) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 3-fold lower expression of the protein Immunoglobulin heavy chain - variable region (fragment) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
U koraku utvrđivanja razlike između proteomskog profila uzorka i proteomskog profila kontrole, utvrđena razlika se očituje u 3 puta manjoj ekspresiji proteina Vitamin D vezujućeg proteina, varijacija (fragment) kod postojanja bolesti deficijencije SAHH u uzorku u odnosu na njegovu ekspresiju u kontroli. In the step of determining the difference between the proteomic profile of the sample and the proteomic profile of the control, the determined difference is manifested in a 3-fold lower expression of the protein Vitamin D binding protein, a variation (fragment) in the presence of SAHH deficiency disease in the sample compared to its expression in the control.
Kako bi se proteomski profil u uzorku krvne plazme mogao uspoređivati s kontrolnim proteomskim profilom karakterističnim za deficijenciju SAHH, potrebno je proteine u ispitivanom uzorku prije svega razdvojiti. Za razdvajanje proteina mogu se koristiti standardne metode kvantitativno-kvalitativne analize proteina. Jedna od poželjnih standardnih metoda analize proteina je metoda gel elektroforeze (2-DE) u kombinaciji sa spektrometrijom masa. Postoji cijeli niz drugih standardnih metoda analize proteina kao što su kapilarna elektroforeza, tekućinska kromatografija, ionska kromatografija i sl. također u kombinaciji sa spektrometrijom masa. In order to compare the proteomic profile in the blood plasma sample with the control proteomic profile characteristic of SAHH deficiency, it is first necessary to separate the proteins in the tested sample. Standard methods of quantitative-qualitative protein analysis can be used for protein separation. One of the preferred standard methods of protein analysis is the gel electrophoresis (2-DE) method in combination with mass spectrometry. There are a whole range of other standard methods of protein analysis such as capillary electrophoresis, liquid chromatography, ion chromatography, etc. also in combination with mass spectrometry.
Nakon razdvajanja proteina u krvnoj plazmi, potrebno je izvršiti kvantitativnu i kvalitativnu analizu gelova kako bi se utvrdio proteomski profil iz uzorka krvne plazme. After the separation of the proteins in the blood plasma, it is necessary to perform quantitative and qualitative analysis of the gels in order to determine the proteomic profile from the blood plasma sample.
Naime, potrebno je denzitometrijski obraditi gelove gdje se na svakome gelu točno odredi pozicija zacrnjenja (kao pojedinačnog proteina, kvalitativna analiza) i intenzitet samog zacrnjenja (kvantitativna analiza). Metode i uređaji koje se za takvu analizu koriste su poznati osobama iz stručnog područja predmetnog izuma. Namely, it is necessary to process the gels densitometrically, where the position of the blackening (as an individual protein, qualitative analysis) and the intensity of the blackening itself (quantitative analysis) are determined exactly on each gel. The methods and devices used for such analysis are known to persons skilled in the field of the subject invention.
Kod nekih od gore navedenih metoda analize proteina, preciznije, kod metoda koje koriste izoelektrično fokusiranje, poželjno je da se analiza proteina u krvnoj plazmi napravi u dva različita IPG područja, odnosno dva različita pH raspona, radi dobivanja što jasnije međusobne razdvojenosti proteina na gelu. Analiza proteina u krvnoj plazmi bi se prema tome trebala napraviti dva puta, koristeći istu metodu analize s tim da se kod jedne analize proteina koriste IPG trake raspona pH 3-10 a da se kod druge analize koriste IPG trake raspona pH 4-7. Na taj način se dobiju jasni podaci o ekspresiji svih gore naznačenih proteina u uzorku. Takav način ispitivanja uzorka krvne plazme je poželjan jer daje najdetaljnije podatke o kvantitativnom i kvalitativnom profilu ekspresije proteina koji se kod oboljelih od deficijencije SAHH razlikuju od kontrole. In some of the above-mentioned methods of protein analysis, more precisely, in the methods that use isoelectric focusing, it is desirable that the analysis of proteins in blood plasma be done in two different IPG areas, i.e. two different pH ranges, in order to obtain the clearest separation of proteins on the gel. The analysis of proteins in blood plasma should therefore be done twice, using the same method of analysis, with one protein analysis using IPG strips in the pH range 3-10 and the second analysis using IPG strips in the pH range 4-7. In this way, clear data on the expression of all the above-mentioned proteins in the sample is obtained. Such a method of testing a blood plasma sample is desirable because it provides the most detailed data on the quantitative and qualitative expression profile of proteins that are different in patients with SAHH deficiency compared to controls.
Preciznije, upotrebom pH raspona pH 3-10 kod metode razdvajanja proteina u uzorku, dobio se jasan proteomski profil za sljedeće proteine: Apolipoprotein A-IV (prekursor), Fikolin-3 (prekursor), Serotransferin (prekursor), Transtiretin (prekursor), Haptoglobin (prekursor), Cink alfa 2 glikoprotein (prekursor). More precisely, using the pH range pH 3-10 in the protein separation method in the sample, a clear proteomic profile was obtained for the following proteins: Apolipoprotein A-IV (precursor), Ficolin-3 (precursor), Serotransferrin (precursor), Transthyretin (precursor), Haptoglobin (precursor), Zinc alpha 2 glycoprotein (precursor).
Nadalje, upotrebom pH raspona pH 4-7 kod metode razdvajanja proteina u uzorku, dobio se jasan proteomski profil za sljedeće proteine: LOMP protein, Vitamin D vezujući protein prekursor, Apolipoprotein A-I (fragment), Imunoglobulin teški lanac - varijabilna regija (fragment), Vitamin D vezujući protein, varijacija (fragment), Fosfotirozil fosfatazni aktivator, T-stanični receptor delta lanac HE/801. Furthermore, using the pH range pH 4-7 in the protein separation method in the sample, a clear proteomic profile was obtained for the following proteins: LOMP protein, Vitamin D binding protein precursor, Apolipoprotein A-I (fragment), Immunoglobulin heavy chain - variable region (fragment), Vitamin D binding protein, variation (fragment), Phosphotyrosyl phosphatase activator, T-cell receptor delta chain HE/801.
Kratak opis slika Short description of the pictures
Slika 1 - proteomski profil plazme kontrole na pH 3-10 Figure 1 - proteomic profile of control plasma at pH 3-10
Slika 2 - proteomski profil plazme uzorka pacijenta na pH 3-10. Figure 2 - proteomic profile of patient plasma sample at pH 3-10.
Slika 3 – proteomski profil plazme kontrole na pH 4-7 Figure 3 – proteomic profile of control plasma at pH 4-7
Slika 4 – proteomski profil plazme uzorka pacijenta na pH 4-7 Figure 4 – proteomic profile of patient sample plasma at pH 4-7
Slike 1 i 2 pokazuju primjere proteomskog profila plazme dobivenog uz pomoć metode 2-DE. Izoelektrično fokusiranje je provedeno s ukupno 120 µg proteina po traci pH raspona 3-10 NL, dok je vertikalna poliakrilamidna elektroforeza provedena na 12% gelu veličine 20 x 18 cm. Gelovi su obojani fluorescentnom bojom SYPRO Ruby. Proteomski profil kontrole, koji se vidi na Slici 1 predstavlja jasne profile ekspresije sljedećih proteina: Apolipoprotein A-IV (prekursor), Fikolin-3 (prekursor), Serotransferin (prekursor), Transtiretin (prekursor), Haptoglobin (prekursor). Proteomski profil uzorka, koji se vidi na slici 2 predstavlja profile ekspresije sljedećih proteina: Apolipoprotein A-IV (prekursor), Fikolin-3 (prekursor), Serotransferin (prekursor), Transtiretin (prekursor), Cink alfa 2 glikoprotein (prekursor). Uspoređujući slike 1 i 2 jasno se vidi razlika u profilu ekspresije proteina između kontrole i uzorka pacijenta. Figures 1 and 2 show examples of the plasma proteomic profile obtained using the 2-DE method. Isoelectric focusing was performed with a total of 120 µg of protein per lane in the pH range 3-10 NL, while vertical polyacrylamide electrophoresis was performed on a 12% gel of size 20 x 18 cm. The gels were stained with fluorescent dye SYPRO Ruby. The proteomic profile of the control, which can be seen in Figure 1, presents clear expression profiles of the following proteins: Apolipoprotein A-IV (precursor), Ficolin-3 (precursor), Serotransferrin (precursor), Transthyretin (precursor), Haptoglobin (precursor). The proteomic profile of the sample, which can be seen in Figure 2, represents the expression profiles of the following proteins: Apolipoprotein A-IV (precursor), Ficolin-3 (precursor), Serotransferrin (precursor), Transthyretin (precursor), Zinc alpha 2 glycoprotein (precursor). Comparing Figures 1 and 2 clearly shows the difference in the protein expression profile between the control and the patient sample.
Slike 3 i 4 pokazuju primjere proteomskog profila plazme dobivenog pomoću metode 2-DE. Izoelektrično fokusiranje provedeno je s ukupno 250 µg proteina po traci pH raspona 4-7 NL, dok je vertikalna poliakrilamidna elektroforeza provedena na 12% gelu veličine 20 x 18 cm. Gelovi su obojani fluorescentnom bojom SYPRO Ruby. Proteomski profil kontrole, koji se vidi na Slici 3 predstavlja jasne profile ekspresije sljedećih proteina: LOMP protein, Vitamin D vezujući protein prekursor, Apolipoprotein A-I (fragment), Imunoglobulin teški lanac - varijabilna regija (fragment), Vitamin D vezujući protein, varijacija (fragment), Fosfotirozil fosfatazni aktivator, T-stanični receptor delta lanac HE/801. Proteomski profil uzorka, koji se vidi na Slici 4 predstavlja profile ekspresije sljedećih proteina: LOMP protein, Vitamin D vezujući protein prekursor, Apolipoprotein A-I (fragment), Imunoglobulin teški lanac - varijabilna regija (fragment), Vitamin D vezujući protein, varijacija (fragment), Fosfotirozil fosfatazni aktivator, T-stanični receptor delta lanac HE/801. Uspoređujući slike 3 i 4 jasno se vidi razlika u profilu ekspresije proteina između kontrole i uzorka pacijenta. Figures 3 and 4 show examples of the plasma proteomic profile obtained using the 2-DE method. Isoelectric focusing was performed with a total of 250 µg of protein per lane in the pH range 4-7 NL, while vertical polyacrylamide electrophoresis was performed on a 12% gel of size 20 x 18 cm. The gels were stained with fluorescent dye SYPRO Ruby. The control proteomic profile, seen in Figure 3, presents clear expression profiles of the following proteins: LOMP protein, Vitamin D binding protein precursor, Apolipoprotein A-I (fragment), Immunoglobulin heavy chain - variable region (fragment), Vitamin D binding protein, variation (fragment ), Phosphotyrosyl phosphatase activator, T-cell receptor delta chain HE/801. The proteomic profile of the sample, which can be seen in Figure 4, represents the expression profiles of the following proteins: LOMP protein, Vitamin D binding protein precursor, Apolipoprotein A-I (fragment), Immunoglobulin heavy chain - variable region (fragment), Vitamin D binding protein, variation (fragment) , Phosphotyrosyl phosphatase activator, T-cell receptor delta chain HE/801. Comparing Figures 3 and 4 clearly shows the difference in the protein expression profile between the control and the patient sample.
Detaljan opis najmanje jednog od načina ostvarivanja izuma A detailed description of at least one way of realizing the invention
U jednom od poželjnih načina ostvarivanja predmetnog izuma, koristio se glavni metodološki pristup u proteomskim istraživanjima, odnosno klasični pristup uz pomoć metode dvodimenzionalne gel-elektroforeze (2-DE) i pristup bez gela tzv. “shotgun” pristup. (Shaw, M.M.;Riederer, B.M. Proteomics, 2003, 3, 1408.; Garfin, D.E. Trac-Trends Anal. Chem, 2003, 22, 263.) Metoda 2-DE koristi se za razdvajanje složenih proteinskih smjesa pri čemu se odabrani proteini identificiraju uz pomoć spektrometrije masa. Shotgun metoda temelji se na direktnoj analizi proteina izdvojenih digestijom iz složenih proteinskih smjesa uz pomoć tekućinske kromatografije/masene spektrometrije (LC-MS). Kod klasičnog pristupa analize proteinskih smjesa uz pomoć gelova koriste se imobilizirani gelovi s gradijentom pH u kojima se proteini razdvajaju na temelju njihove izoelektrične točke nakon čega slijedi razdvajanje proteina uz pomoć okomite, SDS elektroforeze pri čemu se proteini razdvajaju na temelju njihove molekulske mase. In one of the preferred ways of realizing the subject invention, the main methodological approach in proteomic research was used, i.e. the classic approach with the help of the two-dimensional gel-electrophoresis (2-DE) method and the so-called gel-free approach. "shotgun" approach. (Shaw, M.M.; Riederer, B.M. Proteomics, 2003, 3, 1408.; Garfin, D.E. Trac-Trends Anal. Chem, 2003, 22, 263.) The 2-DE method is used to separate complex protein mixtures in which selected proteins identified with the help of mass spectrometry. The shotgun method is based on the direct analysis of proteins separated by digestion from complex protein mixtures with the help of liquid chromatography/mass spectrometry (LC-MS). The classic approach to analyzing protein mixtures using gels uses immobilized pH gradient gels in which proteins are separated based on their isoelectric point, followed by vertical, SDS electrophoresis, where proteins are separated based on their molecular weight.
Ovisno o veličini gela, korištenom gradijentu pH i metodi bojanja gela, uz pomoć dvodimenzionalne elektroforeze može se istovremeno razdvojiti više od 5000 proteina (~2000 proteina rutinski), odnosno može se detektirati i kvantificirati < 1 ng proteina po proteinskoj točki (engl. spot; Gorg i sur. 2004). Svaka proteinska točka predstavlja različiti protein, različitu izoformu istog proteina ili njegovu poslijetranslacijsku modifikaciju [Sivakumar, A. (2002) 2D gels and bioinformatics--an eye to the future. In Silico Biol 2, 507-510, 2002]. Analizom gelova dobivaju se proteomski profili ekspresije na temelju kojih se određuju promijene u ekspresiji proteina. Kvalitativna i kvantitativna analiza 2-DE gelova vrši se uz pomoć specijaliziranih komercijalnih programa poput Melanie, PDQuest, Z3 i Z4000, Phoretix i Progenesis (Marengo E. et al. (2005) Numerical approaches for quantitative analysis of two-dimensional maps: A review of commercial software and home-made systems. Proteomics 5: 654-666). Depending on the size of the gel, the pH gradient used and the gel staining method, with the help of two-dimensional electrophoresis, more than 5000 proteins can be separated simultaneously (~2000 proteins routinely), that is, < 1 ng of protein per protein spot can be detected and quantified. Gorg et al. 2004). Each protein spot represents a different protein, a different isoform of the same protein or its post-translational modification [Sivakumar, A. (2002) 2D gels and bioinformatics--an eye to the future. In Silico Biol 2, 507-510, 2002]. By analyzing the gels, proteomic expression profiles are obtained, on the basis of which changes in protein expression are determined. Qualitative and quantitative analysis of 2-DE gels is performed with the help of specialized commercial programs such as Melanie, PDQuest, Z3 and Z4000, Phoretix and Progenesis (Marengo E. et al. (2005) Numerical approaches for quantitative analysis of two-dimensional maps: A review of commercial software and home-made systems.Proteomics 5: 654-666).
Metodološki protokol po kojemu su uzorci ispitivani osigurava reproducibilnost rezultata. The methodological protocol by which the samples were tested ensures the reproducibility of the results.
Osnovne kemikalije Basic chemicals
C3H6O, 100 %, mineralno ulje, Tris-HCl, urea, C3H8O3, SDS, DTT, IAA, akrilamid, minimum 99 %, bisakrilamid, minimum 98 %, (NH4)2S2O8, 98+ %, TEMED, n-butanol, agaroza, C2H5OH, CH3COOH, H3PO4, (NH4)2SO4, CH3OH, albumin goveđeg seruma, C2H5NO2, minimum 98 %, tripsin, NaHCO3 lužnata otopina bakrenog tartarata (reagens A'), Folin-Ciocalteuov reagens (reagens B'), CHCA, MeCN, HCOOH, TFA, triton X-100, CHAPSTM (Sigma, SAD), BIO-LYTETM AMFOLITE 3-10 (BIO-RAD, SAD), tiourea, NH4HCO3. C3H6O, 100%, mineral oil, Tris-HCl, urea, C3H8O3, SDS, DTT, IAA, acrylamide, minimum 99%, bisacrylamide, minimum 98%, (NH4)2S2O8, 98+%, TEMED, n-butanol, agarose , C2H5OH, CH3COOH, H3PO4, (NH4)2SO4, CH3OH, bovine serum albumin, C2H5NO2, minimum 98%, trypsin, NaHCO3 alkaline solution of copper tartrate (reagent A'), Folin-Ciocalteu's reagent (reagent B'), CHCA, MeCN , HCOOH, TFA, triton X-100, CHAPSTM (Sigma, USA), BIO-LYTETM AMFOLITE 3-10 (BIO-RAD, USA), thiourea, NH4HCO3.
Osnovne puferske otopine Basic buffer solutions
Korišteni su puferi: pufer za elektroforezu (25 mM Tris, 192 mM glicin, 0,1 % SDS, pH=8,3), ekvilibarcijski pufer (20 mM Tris-HCl, pH 8,8, 6 M urea, 30 % w/v glicerol, 2 % w/v SDS, bromfenol modrilo), pufer za rehidrataciju IPG-traka pH raspona 4-7 (9 M urea, 4 % (w/v) CHAPSTM, 0,5 % (v/v) TRITONTM X-100, 0,2 /% (w/v) smjesa amfolita BIO-LYTETM AMFOLIT pH 3-10, 1 % (w/v) DTT), te pufer za rehidrataciju IPG-traka pH raspona 3-10 (7 M urea, 2 M tiourea, 4 % (w/v) CHAPSTM, 0,2 /% (w/v) smjesa amfolita BIO-LYTETM AMFOLIT pH 3-10, 1 % (w/v) DTT). Buffers were used: electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1 % SDS, pH=8.3), equilibration buffer (20 mM Tris-HCl, pH 8.8, 6 M urea, 30 % w /v glycerol, 2% w/v SDS, bromphenol blue), buffer for rehydration of IPG-strips pH range 4-7 (9 M urea, 4% (w/v) CHAPSTM, 0.5% (v/v) TRITONTM X-100, 0.2 /% (w/v) ampholyte mixture BIO-LYTETM AMPHOLIT pH 3-10, 1 % (w/v) DTT), and buffer for rehydration of IPG-strips pH range 3-10 (7 M urea, 2 M thiourea, 4 % (w/v) CHAPSTM, 0.2 /% (w/v) ampholyte mixture BIO-LYTETM AMPHOLIT pH 3-10, 1 % (w/v) DTT).
Boje Colors
Korištene su sljedeće boje: Comassie Brilliant Blue G-250, 0,1 % (w/v) (BIO-RAD, SAD), Tris (bathophenatroline disulfonate)-ruthenium (II) sodium salt solution (Biochemika, Fluka, Sigma Aldrich, SAD) te Bromphenol Blue, 0,0002 % (w/v) (Fluka, Sigma, SAD). The following colors were used: Comassie Brilliant Blue G-250, 0.1 % (w/v) (BIO-RAD, USA), Tris (bathophenatroline disulfonate)-ruthenium (II) sodium salt solution (Biochemika, Fluka, Sigma Aldrich, USA) and Bromphenol Blue, 0.0002 % (w/v) (Fluka, Sigma, USA).
Ostali materijali Other materials
Korišteni su komercijalni paketi za pročišćavanje uzoraka krvne plazme- Aurum™ Serum Protein Mini Kit (BIO-RAD, SAD), te za kvantifikaciju proteina- DC Protein Assay Kit (BIO-RAD, SAD). Commercial packages were used for purification of blood plasma samples - Aurum™ Serum Protein Mini Kit (BIO-RAD, USA), and for protein quantification - DC Protein Assay Kit (BIO-RAD, USA).
Tehnička oprema Technical equipment
U ovom načinu ostvarivanja predmetnog izuma, korištena je sljedeća oprema: centrifuga 5415 R (Eppendorf, Švicarska), mikrotitarska pločica (Beckton Dickinson Labware, SAD), IPG-trake (17 cm, pH 3-10 NL i 17 cm, pH 4-7 N; BIO-RAD, SAD), PROTEAN IEF cell (BIO-RAD, SAD), jedinica za hlađenje sustava za vertikalnu elektroforezu (PROTEAN II XL cell; BIO-RAD, SAD), Versa Doc Imaging System, model 4000 (BIO-RAD, SAD), PDQuest SW, verzija 7,0 (BIO-RAD, SAD), ZipTip (C4, Millipore, SAD), spektrofotometar (Thermolab Systems, SAD), vakuum centrifuga (Eppendorf, Švicarska), MALDI pločica (Applied Biosystems, SAD), MALDI-TOF spektrometar masa, model Voyager DE STR Biospectrometry workstation (Applied Biosystems, SAD), Mascot verzija 1.9.05 (Matrix Science, Velika Britanija). In this way of realizing the subject invention, the following equipment was used: centrifuge 5415 R (Eppendorf, Switzerland), microtiter plate (Beckton Dickinson Labware, USA), IPG strips (17 cm, pH 3-10 NL and 17 cm, pH 4- 7 N; BIO-RAD, USA), PROTEAN IEF cell (BIO-RAD, USA), vertical electrophoresis system cooling unit (PROTEAN II XL cell; BIO-RAD, USA), Versa Doc Imaging System, model 4000 (BIO -RAD, USA), PDQuest SW, version 7.0 (BIO-RAD, USA), ZipTip (C4, Millipore, USA), spectrophotometer (Thermolab Systems, USA), vacuum centrifuge (Eppendorf, Switzerland), MALDI plate (Applied Biosystems, USA), MALDI-TOF mass spectrometer, model Voyager DE STR Biospectrometry workstation (Applied Biosystems, USA), Mascot version 1.9.05 (Matrix Science, Great Britain).
Prilikom poželjnog načina izvođenja predmetnog izuma, korišten je Aurum™ Serum Protein Mini Kit za uklanjanje najzastupljenijih serumskih proteina u krvnoj plazmi, a isti se koristio prema uputama proizvođača. Nakon završenog postupka uklanjanja najzastupljenijih serumskih proteina, u epruveti se dobiva uzorak krvne plazme pročišćen od IgG i albumina koji je pripremljen za analizu na 2-DE gelu. In the preferred way of carrying out the subject invention, the Aurum™ Serum Protein Mini Kit was used to remove the most abundant serum proteins in blood plasma, and it was used according to the manufacturer's instructions. After the removal of the most abundant serum proteins, a blood plasma sample purified from IgG and albumin is obtained in a test tube and prepared for analysis on a 2-DE gel.
Koncentracija proteina u lizatu određena je uz pomoć komercijalno dostupnog kompleta za kvantifikaciju proteina (DC Protein Assay Kit). Lizati za izoelektrično fokusiranje pripremaju se taloženjem acetonom, pri čemu se uklanjaju ionske nečistoće i lipidi te se proteini istovremeno koncentriraju. Proteinski talog se potom otopi zagrijavanjem na 30 °C u 300-330 µL pufera za rehidrataciju IPG traka, koji je po svom sastavu identičan upotrijebljenom 2-DE puferu za lizu stanica, ali ne sadrži smjesu nukleaza niti inhibitore proteaza. Tako otopljenom uzorku se dodaje bromfenol modrilo, kratko se promiješa i centrifugira radi uklanjanja svih neotopljenih čestica. Potom se na uzorak pažljivo položi IPG traka (17 cm, pH 3-10 i pH 4-7). Nakon toga se ispod svake trake na oba njezina kraja stave navlaženi filter-papiri, te se dalje provodi izoelektrično fokusiranje. Nakon završetka fokusiranja, IPG trake se ocijede od mineralnog ulja te se provodi tzv. ekvilibracija IPG traka, čime se one pripremaju za drugu dimenziju. Protein concentration in the lysate was determined using a commercially available protein quantification kit (DC Protein Assay Kit). Lysates for isoelectric focusing are prepared by acetone precipitation, whereby ionic impurities and lipids are removed and proteins are simultaneously concentrated. The protein precipitate is then dissolved by heating at 30 °C in 300-330 µL buffer for rehydration of IPG strips, which in its composition is identical to the used 2-DE buffer for cell lysis, but does not contain a mixture of nucleases or protease inhibitors. Bromphenol blue is added to the dissolved sample, mixed briefly and centrifuged to remove all undissolved particles. An IPG strip (17 cm, pH 3-10 and pH 4-7) is then carefully placed on the sample. After that, moistened filter papers are placed under each strip at both ends, and isoelectric focusing is then carried out. After focusing, the IPG strips are drained of mineral oil and the so-called equilibration of the IPG bands, which prepares them for the second dimension.
Pri tome se trake urone u epruvete s ekvilibracijskim puferom kojem je dodana 1 % otopina w/v ditiotreitola (DTT). Nakon toga, trake se prebace u nove epruvete s ekvilibracijskim puferom kojem je dodana 2,5 % otopina jodoacetamida (IAA). Tako pripremljene trake su spremne za slijedeću dimenziju razdvajanja proteina, SDS gel elektroforezu. In doing so, the strips are immersed in test tubes with an equilibration buffer to which a 1% w/v solution of dithiothreitol (DTT) has been added. After that, the strips are transferred to new test tubes with equilibration buffer to which 2.5% iodoacetamide (IAA) solution was added. Strips thus prepared are ready for the next dimension of protein separation, SDS gel electrophoresis.
SDS-PAGE SDS-PAGE
Elektroforeza je vođena pri denaturirajućim uvjetima za što je korišten tzv. Laemmlijev tris-glicin puferski sistem (Laemmli 1970). Za analizu su korišteni 12 % gelovi debljine 1 mm i dimenzija 20 x 18 cm koji su pripremljeni od 33,5 mL destilirane vode, 25 mL 1,5 M Tris-HCl pH 8,8, 1 mL 10 % (w/v) SDS, 40 mL otopine akrilamid/bisakrilamid (30,8 % T; 2,7 % C bis), 500 µL 10 % (w/v) amonijevog persulfata i 50 µL TEMED-a. Nakon što se gelovi polimeriziraju, uklanja se n-butanol, te se površina gelova ispire nekoliko puta s destiliranom vodom. Staklene ploče s gelovima se potom umeću u jedinicu za hlađenje sustava za vertikalnu elektroforezu (PROTEAN II XL cell) i prebace u komoru s puferom za elektroforezu (25 mM Tris, 192 mM glicin, 0,1 % SDS, pH 8,3). Prethodno ekvilibrirane trake se jednokratno isperu kratkim uranjanjem u pufer za elektroforezu, pažljivo umetnu između stakala. Potom se trake preliju s 0,5 %-tnom otopinom agaroze koja se pripremi otapanjem 0,5 g agaroze i 200 µl 0,0002 % (w/v) bromfenol modrila u 100 mL pufera za elektroforezu. Kada se agaroza skrutne, u gornju se komoru izlije pufer za elektroforezu, jedinica za hlađenje se spoji na vodu, stavi se poklopac i započne elektroforeza. Electrophoresis was conducted under denaturing conditions, for which the so-called Laemmli's tris-glycine buffer system (Laemmli 1970). For the analysis, 12% gels with a thickness of 1 mm and dimensions of 20 x 18 cm were used, which were prepared from 33.5 mL of distilled water, 25 mL of 1.5 M Tris-HCl pH 8.8, 1 mL of 10 % (w/v) SDS, 40 mL acrylamide/bisacrylamide solution (30.8 % T; 2.7 % C bis), 500 µL 10 % (w/v) ammonium persulfate and 50 µL TEMED. After the gels are polymerized, the n-butanol is removed, and the surface of the gels is washed several times with distilled water. Glass plates with gels are then inserted into the cooling unit of the vertical electrophoresis system (PROTEAN II XL cell) and transferred to a chamber with electrophoresis buffer (25 mM Tris, 192 mM glycine, 0.1% SDS, pH 8.3). Previously equilibrated strips are washed once by short immersion in electrophoresis buffer, carefully inserted between glass slides. The strips are then covered with a 0.5% agarose solution, which is prepared by dissolving 0.5 g of agarose and 200 µl of 0.0002% (w/v) bromophenol blue in 100 mL of electrophoresis buffer. When the agarose has solidified, electrophoresis buffer is poured into the upper chamber, the cooling unit is connected to water, the lid is put on and electrophoresis is started.
Snimanje i analiza gelova Imaging and analysis of gels
Nakon bojanja SYPRO Ruby-em gelovi su slikani uz pomoć uređaja za slikanje (VersaDoc Imaging System, model 4000) pri vremenu ekspozicije od 25 i 30 sekundi. Kvalitativana i kvantitativna analiza gelova je provedena pomoću specijalnog računalnog programa za analizu 2-DE gelova (PDQuest SW, verzija 7,0). Prije analize gelovi su normalizirani tzv. metodom ukupne gustoće na slici gela (engl. total density in gel image), kod koje se zbrajaju vrijednosti optičkih gustoća svih točaka unutar slike. Usporedba tih zbrojenih vrijednosti služi kao osnova za normalizaciju. Kao signifikantna vrijednost uzeta je tri puta (za 2-DE gelove sa uzorcima fokusiranim na IPG trakama pH raspona 4-7) i pet puta (za 2-DE gelove sa uzorcima fokusiranim na IPG trakama pH raspona 3-10) povećana ekspresija u plazmi pacijenta. After staining with SYPRO Ruby, the gels were imaged using an imaging device (VersaDoc Imaging System, model 4000) at exposure times of 25 and 30 seconds. Qualitative and quantitative analysis of the gels was performed using a special computer program for the analysis of 2-DE gels (PDQuest SW, version 7.0). Before the analysis, the gels were normalized by the so-called using the total density in gel image method, where the optical density values of all points within the image are added up. The comparison of these summed values serves as the basis for normalization. Three times (for 2-DE gels with samples focused on IPG bands in the pH range 4-7) and five times (for 2-DE gels with samples focused on IPG bands in the pH range 3-10) increased expression in plasma was taken as a significant value. the patient.
Dobiveni rezultati poželjnog načina izvođenja predmetnog izuma, uspoređivani su sa kontrolnim proteomskim profilom te su se utvrdile razlike koje ukazuju na deficijenciju SAHH u uzorku a koje se nalaze u Tablicama 1 i 2. Na taj način se utvrdilo da li ispitivana osoba ima deficijenciju SAHH. The obtained results of the preferred way of carrying out the subject invention were compared with the control proteomic profile, and the differences indicating SAHH deficiency in the sample were determined, which are found in Tables 1 and 2. In this way, it was determined whether the examined person has SAHH deficiency.
Tablica 1. Lista diferencijalno eksprimiranih proteina u plazmi pacijenata oboljelih od SAHH deficijencije identificiranih pomoću MALDI-TOF spektrometra masa, a koji su dobiveni analizom 2-DE uz pomoć IPG traka raspona pH 3-10 NL. Table 1. List of differentially expressed proteins in the plasma of patients suffering from SAHH deficiency identified by MALDI-TOF mass spectrometer, which were obtained by 2-DE analysis with the help of IPG bands in the range of pH 3-10 NL.
[image] [image]
Tablica 2. Lista diferencijalno eksprimiranih proteina identificiranih u plazmi pacijenata oboljelih od SAHH deficijencije pomoću MALDI-TOF spektrometra masa, a koji su dobiveni analizom 2-DE uz pomoć traka IPG raspona pH 4-7. Table 2. List of differentially expressed proteins identified in the plasma of patients suffering from SAHH deficiency using MALDI-TOF mass spectrometer, which were obtained by 2-DE analysis with the help of IPG strips in the pH range 4-7.
[image] [image]
Ispitivanje gore navedenog poželjnog načina izvođenja predmetnog izuma Examination of the above-mentioned preferred way of carrying out the subject invention
S obzirom da je bolest deficijencije SAHH vrlo rijetka, ispitivanja su se mogla vršiti na malom broju pacijenata te u istom broju kontrolnih zdravih osoba. Kontrolne zdrave osobe su bile osobe koje su metabolički zdrave osobe. Naime, ispitivanja su rađena na tri oboljele osobe od deficijencije SAHH te na tri metabolički zdrave osobe. Ispitivanja su vršena na način da je svaki par oboljela-kontrolna zdrava osoba bila iste životne dobi (npr. oboljela osoba stara 15 dana – kontrolna zdrava osoba stara 15 dana). Given that SAHH deficiency disease is very rare, tests could be performed on a small number of patients and the same number of control healthy individuals. Control healthy people were people who are metabolically healthy people. Namely, the tests were performed on three people suffering from SAHH deficiency and on three metabolically healthy people. The tests were performed in such a way that each pair of a sick person and a healthy control person was of the same age (eg a sick person 15 days old - a healthy control person 15 days old).
Ispitivanje se radilo na način da bi se uzorak krvi pacijenta i kontrolni uzorak krvi zdrave osobe tretira gore navedenom metodom analize uzorka kako bi se utvrdio proteomski profil oba uzorka. The test was done in such a way that the blood sample of the patient and the control blood sample of a healthy person would be treated with the above sample analysis method in order to determine the proteomic profile of both samples.
Proteomski profili bolesnih osoba su pokazali preklapanje s proteomskim profilima prikazanim u tabeli 1 i 2, dok kod proteomskih profila kontrolnih zdravih osoba nije ustanovljena analogija s tabelama 1 i 2 tj. nije bilo odstupanja od standardnog proteomskog profila osobe koja nije oboljela od deficijencije SAHH. The proteomic profiles of the sick persons showed an overlap with the proteomic profiles shown in Tables 1 and 2, while the proteomic profiles of the control healthy persons were not analogous to Tables 1 and 2, i.e. there were no deviations from the standard proteomic profile of a person who did not suffer from SAHH deficiency.
Način primjene izuma Method of application of the invention
Predmetni izum prema tome, omogućava neinvazivno dijagnosticiranje deficijencije SAAH iz krvne plazme. The present invention therefore enables non-invasive diagnosis of SAAH deficiency from blood plasma.
Pored dijagnosticiranja postojanja bolesti u osoba, predmetni izum se može koristiti i za praćenje promjena u ekspresiji proteina nakon provedene terapije, odnosno praćenje uspješnosti terapije. In addition to diagnosing the existence of a disease in a person, the subject invention can also be used to monitor changes in protein expression after the therapy, i.e. monitoring the success of the therapy.
Predmetni izum pruža brzu, jeftinu i pouzdanu dijagnostičku metodu za dijagnosticiranje i praćenje bolesti deficijencije, tako da se predmetna metoda pored u medicinske svrhe, može koristiti i kao kontrolna metoda u ispitivanju metaboličkih poremećaja kod oboljelih od deficijencije SAHH. The subject invention provides a fast, cheap and reliable diagnostic method for diagnosing and monitoring deficiency diseases, so that the subject method, in addition to medical purposes, can also be used as a control method in examining metabolic disorders in patients with SAHH deficiency.
Predmetni izum se također može koristiti u svrhu pronalaženja terapije za bolest deficijencija SAAH. The subject invention can also be used for the purpose of finding a therapy for SAAH deficiency disease.
Claims (22)
Priority Applications (2)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HR20080563A HRP20080563A2 (en) | 2008-11-05 | 2008-11-05 | Proteomics profiles and detection methods of proteins in blood plasma |
| PCT/HR2009/000030 WO2010052510A1 (en) | 2008-11-05 | 2009-11-04 | Proteomics profiles and detection methods of proteins in blood plasma for the diagnosis of sahh deficiency |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| HR20080563A HRP20080563A2 (en) | 2008-11-05 | 2008-11-05 | Proteomics profiles and detection methods of proteins in blood plasma |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| HRP20080563A2 true HRP20080563A2 (en) | 2010-08-31 |
Family
ID=41785921
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| HR20080563A HRP20080563A2 (en) | 2008-11-05 | 2008-11-05 | Proteomics profiles and detection methods of proteins in blood plasma |
Country Status (2)
| Country | Link |
|---|---|
| HR (1) | HRP20080563A2 (en) |
| WO (1) | WO2010052510A1 (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP3187180A1 (en) * | 2015-12-28 | 2017-07-05 | Ludwig-Maximilians-Universität München | Adenosylhomocysteinase binding substances for medical use |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| ITRM20050085A1 (en) * | 2005-02-28 | 2006-09-01 | Angela Maria Amorini | METHOD FOR THE SIMULTANEOUS DETERMINATION OF PURINE AND PYRIMIDINE DERIVATIVES, N-ACETYLATED AMINO ACIDS AND DICARBOSSYLIC ACIDS IN A BIOLOGICAL SAMPLE BY LIQUID CHROMATOGRAPHY AT HIGH PERFORMANCE (HPLC) AND ITS KIT. |
-
2008
- 2008-11-05 HR HR20080563A patent/HRP20080563A2/en not_active Application Discontinuation
-
2009
- 2009-11-04 WO PCT/HR2009/000030 patent/WO2010052510A1/en active Application Filing
Also Published As
| Publication number | Publication date |
|---|---|
| WO2010052510A1 (en) | 2010-05-14 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Hirsch et al. | Proteomics: current techniques and potential applications to lung disease | |
| Puchades et al. | Proteomic studies of potential cerebrospinal fluid protein markers for Alzheimer's disease | |
| Yuan et al. | Proteomics analysis of human cerebrospinal fluid | |
| DK2569446T3 (en) | DIAGNOSTIC MARKERS FOR NEUROPsychiatric Disease | |
| Miike et al. | Proteome profiling reveals gender differences in the composition of human serum | |
| Jorge et al. | Statistical Model to Analyze Quantitative Proteomics Data Obtained by 18O/16O Labeling and Linear Ion Trap Mass Spectrometry: Application to the Study of Vascular Endothelial Growth Factor-induced Angiogenesis in Endothelial Cells* S | |
| Laila et al. | Role of proteomics in the discovery of autism biomarkers | |
| EP2444814B9 (en) | Biomarker for mental disorders including cognitive disorders, and method using said biomarker to detect mental disorders including cognitive disorders | |
| KR101946884B1 (en) | Method for diagnosing Behcet's disease by using metabolomics | |
| WO2015014903A2 (en) | Diagnostic tools for alzheimer's disease | |
| US9977036B2 (en) | Diagnostic markers for multiple sclerosis | |
| Sotgia et al. | A diethylpyrocarbonate-based derivatization method for the LC-MS/MS measurement of plasma arginine and its chemically related metabolites and analogs | |
| Hansson et al. | Validation of a prefractionation method followed by two-dimensional electrophoresis–Applied to cerebrospinal fluid proteins from frontotemporal dementia patients | |
| Godzien et al. | Effect of a nutraceutical treatment on diabetic rats with targeted and CE-MS non-targeted approaches | |
| Jacob et al. | Compositional profiling and biomarker identification of the tear film | |
| Squitti et al. | Non-ceruloplasmin copper as a stratification biomarker of Alzheimer’s disease patients: How to measure and use it | |
| KR101806136B1 (en) | Method for diagnosing Behcet's disease with arthritis by using metabolomics | |
| WO2018124235A1 (en) | METHOD FOR DETECTING MITOCHONDRIAL tRNA MODIFICATION | |
| Martín et al. | Proteomics as a new tool in forensic sciences | |
| HRP20080563A2 (en) | Proteomics profiles and detection methods of proteins in blood plasma | |
| Bosque et al. | A 43-kDa TDP-43 species is present in aggregates associated with frontotemporal lobar degeneration | |
| Imai et al. | Comprehensive analysis and comparison of proteins in salivary exosomes of climacteric and adolescent females | |
| Karu et al. | Quantitative proteomic analysis of the central amygdala in neuropathic pain model rats | |
| ABEND et al. | Proteomic analysis of radiation-induced alterations in L929 cells | |
| Petzold | Biomarkers of disease progression |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| A1OB | Publication of a patent application | ||
| ODRP | Renewal fee for the maintenance of a patent |
Payment date: 20101028 Year of fee payment: 3 |
|
| AIPI | Request for the grant of a patent on the basis of a substantive examination of a patent application | ||
| OBST | Application withdrawn |