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METHOD AND SYSTEM FOR ANALYZING N-LINKED SUGAR CHAINS OF GLYCOPROTEIN

外国特許コード F180009395
整理番号 (S2016-1050-N0)
掲載日 2018年4月20日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2017JP029658
国際公開番号 WO 2018034346
国際出願日 平成29年8月18日(2017.8.18)
国際公開日 平成30年2月22日(2018.2.22)
優先権データ
  • 特願2016-161118 (2016.8.19) JP
発明の名称 (英語) METHOD AND SYSTEM FOR ANALYZING N-LINKED SUGAR CHAINS OF GLYCOPROTEIN
発明の概要(英語) Disclosed is a novel means for accurately performing qualitative and quantitative analysis of each of the linkage sites of N-linked sugar chains. In a method for analyzing N-linked sugar chains of glycoprotein according to the present invention, a portion of a glycopeptide-containing sample to be analyzed is treated with endo-β-N-acetylglucosaminidase, sugar chains are cleaved to leave only a single GlcNAc of a chitobiose core on an Asn linked to N-linked sugar chains, the sugar chain-cleaved sample is pre-analyzed using liquid chromatography/mass spectrometry, and the liquid chromatography retention time of the target glycopeptide to be analyzed and m/z of precursor ions are predicted on the basis of the results of the pre-analysis to perform a main analysis. Thereby, the linkage sites and structures of N-linked sugar chains linked to the glycoprotein can be analyzed. By using the sugar chain-cleaved sample as an internal standard in the main analysis, each of the sugar chain linkage sites of sugar chains can also be quantitatively analyzed.
特許請求の範囲(英語) [claim1]
1. N-bound sugar chain fragmenting Glycoproteins obtaining a sample containing a glycopeptide, a glycoprotein fragmentation step, a sugar-containing portion of the sample to react with the end - β-N - acetylglucosaminidase, asparagine (Asn) chain and a coupling portion between the residues of β - 1 chitobiose exists, by cutting the coupling 4, acetyglucosamine N- 1 residues (GlcNAc) (however the GlcNAc is 1 residues of Fucose (Fuc) may be bonded) to cut the sugar chain is left on the peptide, sugar chain-cutting step, a sugar chain is obtained by cutting step to cut the glycoprofile liquid chromatography/mass spectrometry is performed while the peptide sample, the chromatogram, and the product ion spectrum to obtain a mass spectrum, liquid chromatography/mass spectrometry step in advance, Asn GlcNAc residues taking into account the modifications or modified GlcNAc-Fuc de novo sequence search or ion MS/MS for analysis, to determine the position of sugar chain binding in a glycopeptide, sugar chain binding position determining step, liquid chromatography/mass spectrometry and pre-de novo sequence analysis on the result of the search or ion MS/MS, sugar chain before disconnection of the precursor ions and a sugar liquid chromatographic retention time estimate m/z, retention time, m/z estimation step, the remainder of the sample containing the peptide carrierldiluent liquid chromatography/mass spectrometry hexaries, according to a result of the estimation of said holding time and the m/z selected precursor ion peak to be analyzed, with respect to the selected peak mass performs analysis on the selected precursor ions, sugar sugar chain structure is determined, according to the present analysis step, including, a sugar chain binding type N glycoprotein analysis methods.
[claim2]
2. Acetylglucosaminidase, Endo F1,Endo F2,Endo F3,Endo M,Endo H - β-N - end and 1 is selected from one or more Endo S 2 includes one or more of, a method according to claim 1.
[claim3]
3. The sample containing the peptide as the position of the sugar, consisting of an oligosaccharide linked peptides not removed using concentrated sample to a glycopeptide, of claims 1 or 2.
[claim4]
4. Cutting a sugar chain is obtained by the drain step as an internal standard sample glycoprofile cleaving peptide, a sugar-containing sample is added to the remainder of the present analysis step is performed, the termini of the sugar chain binding site of sugar chain further comprising quantitative relative to each other, the method of any one of claims 1-3.
[claim5]
5. As an internal standard The, a sugar chain is a non-cut-cut glycoprofile removing glycopeptide using a peptide sample, of the method according to claim 4.
[claim6]
6. A non-cut position of the sugar on the removal of the sugar chain, and adding acetone to cool, the non-cut position of the sugar chain peptides by separation by precipitation method, or the hydrophilic interaction chromatography using the non-cut assigned to the sugar chain is performed by adsorb and remove the glycopeptide, method according to claim 5.
[claim7]
7. N-linked carbohydrate chain N glycoprotein glycopeptide fragments the prepared from a sample containing, acetylglucosaminidase 1 residues by end - β-N - GlcNAc (however the GlcNAc is 1 residues may be bonded Fuc) in which the peptide residues left on the hair Asn in cut sugar chain sugar chain cleaving peptide for the sample, the chromatogram, acquiring a product ion spectrum and mass spectrum, the pre-liquid chromatography/mass spectrometry data obtaining unit, Asn GlcNAc residues taking into account the modifications or modified GlcNAc-Fuc de novo sequence search or ion MS/MS for analysis, to determine the position of a sugar chain-binding in a sugar, a sugar chain-binding position determination unit and, liquid chromatography/mass spectrometry and pre-de novo sequence analysis based on the result of a search or ion MS/MS, sugar chain before disconnection of the precursor ions and a sugar liquid chromatographic retention time estimate m/z, retention time, m/z and the estimation unit, which is a sample before cutting a sugar chain is present for the analysis of the sample containing the peptide for position of the sugar, present for performing the analysis, the analysis unit that, for the present sample for analysis, the chromatogram, and the estimated hold time to obtain the spectrum of the mass fractions, liquid chromatography/mass spectrometry of the sample for the analysis of the data acquisition unit, in accordance with the result of the estimation of the m/z, the value of the estimated spectrum of the mass fraction of retention time than the selected precursor ion peak to be analyzed, the analysis target peak selection unit, the analysis target peak is selected and the selected precursor ions for mass analysis of the obtained data, determining the sugar chain structure of a peptide, sugar chain structure determination unit, according to the present analysis unit, and to output analyzed results output unit, glycoprotein N N-linked carbohydrate chain analysis system.
[claim8]
8. N-bound sugar chain glycoprofile fragmentation glycoproteins prepared from a sample containing the peptide, 1 residues by end - β-N - acetylglucosaminidase GlcNAc (however the GlcNAc is 1 residues may be bonded Fuc) on the residues in the peptide Asn sugar chain is cut to leave glycoprofile cleaving peptide for the sample, the chromatogram, acquiring a product ion spectrum and mass spectrum, the pre-liquid chromatography/mass spectrometry data obtaining unit, Asn GlcNAc residues taking into account the modified modified or GlcNAc-Fuc de novo sequence search or ion MS/MS for analysis, to determine the position of a sugar chain-binding in a sugar, a sugar chain-binding position determination unit and, liquid chromatography/mass spectrometry and MS/MS pre-de novo sequence analysis on the result of the search or ion, a sugar chain is a sugar before disconnection of the precursor ions and liquid chromatographic retention time estimate m/z, retention time, m/z and the estimation unit, according to the present sample for analysis before disconnection of the sugar chain is a peptide-containing samples, to perform the analysis, the analysis unit configured to, said sugar chain scission peptide sample is added as an internal standard for the present sample for analysis, the chromatogram, and the estimated hold time to obtain a mass spectrum of the fractions, liquid chromatography/mass spectrometry of the sample for the analysis of the data acquisition unit, in accordance with the result of the estimation of the m/z, the value of the estimated hold time of a fraction of the selected precursor ion peak to be analyzed is the mass spectrum, the peak of the analysis target selection unit, the selected analysis target peak of the mass spectrometric data acquired for the selected precursor ions, and to determine the structure of a sugar chain is a sugar, a sugar chain structure determination unit, and the internal standard and each extracted ion chromatograms acquires a sugar, a sugar each internal standard can be determined by calculating the relative intensity of radiation, the termini of the sugar chain binding site of sugar chain relative quantification, quantification unit comprises a sugar chain, this analysis unit and, and to output analyzed results output unit, glycoprotein N N-linked carbohydrate chain analysis system.
[claim9]
9. N glycoprotein for analyzing sugar chain binding type, a 1 or a plurality of computers, a sugar chain binding type N glycoproteins glycoprofile fragmentation is prepared from a sample containing the peptide, 1 residues by end - β-N - acetylglucosaminidase GlcNAc (however the GlcNAc is 1 residues may be bonded Fuc) Asn residues in the peptide to be left on the sugar chain is cut off for the peptide sample cut glycoprofile, chromatogram, acquiring a product ion spectrum and the mass spectrum, liquid chromatography/mass spectrometry data acquisition unit in advance, Asn GlcNAc residues taking into account the modifications or modified GlcNAc-Fuc de novo sequence search or ion MS/MS for analysis, to determine the position of sugar chain binding in a peptide, sugar chain binding position determination unit, liquid chromatography/mass spectrometry and pre-de novo sequence analysis based on the result of a search or ions MS/MS, sugar chain before disconnection of the precursor ions and a sugar liquid chromatographic retention time estimate m/z, retention time, m/z estimation unit, which is a sample for the analysis, a sugar chain by end - β-N - acetylglucosaminidase cutting processing has not been subjected to a peptide-containing samples, for performing the analysis of the present invention, this analysis unit which, for the present sample for analysis, the chromatogram, and the estimated hold time to obtain the spectrum of the mass fractions, liquid chromatography/mass spectrometry of the sample for the analysis of the data acquisition unit, in accordance with the result of the estimation of the m/z, wherein the fraction of the estimated hold time from the mass spectrum of the selected precursor ion peak to be analyzed, the analysis target peak selection unit, and the selected analysis for the selected precursor ions of the target peak of the mass spectrometric data acquired, determining the sugar chain structure of a peptide, sugar chain structure determination unit, the analysis unit, and outputs the result of the analysis program for causing a computer functions as the output unit.
[claim10]
10. N-linked carbohydrate chain N glycoprotein for analysis, one or more computer 1, glycoprotein N N-linked carbohydrate chain glycopeptide fragments the prepared from a sample containing, acetylglucosaminidase 1 residues by end - β-N - GlcNAc (however the GlcNAc is 1 residues may be bonded Fuc) in which the peptide residues left on the hair Asn in cut sugar chain sugar chain cleaving peptide for the sample, the chromatogram, acquiring a product ion spectrum and the mass spectrum, liquid chromatography/mass spectrometry data acquisition unit in advance, Asn GlcNAc residues taking into account the modifications or modified GlcNAc-Fuc de novo sequence search or ion MS/MS for analysis, to determine the position of a sugar chain-binding in a sugar, a sugar chain-binding position determination unit, liquid chromatography/mass spectrometry and pre-de novo sequence analysis on the result of the search or ions MS/MS, sugar chain before disconnection of the precursor ions and a sugar liquid chromatographic retention time estimate m/z, retention time, m/z estimation unit, which is a sample for the analysis peptide-containing sample position of the sugar before cutting a sugar chain, for performing the analysis of the present invention, a present analysis, said sugar chain scission peptide sample is added as an internal standard for the present sample for analysis, the chromatogram, and the estimated hold time to obtain the spectrum of the mass fractions, liquid chromatography/mass spectrometry of the sample for the analysis of the data acquisition unit, in accordance with the result of the estimation of the m/z, wherein the fraction of time that holds the estimated spectrum of the mass to be analyzed are selected from the precursor ion peak, the peak of the analysis target selection unit, the selected analysis target peak of the mass spectrometric data acquired for the selected precursor ions, and to determine the structure of a sugar chain is a sugar, a sugar chain structure determination unit, and the internal standard and each extracted ion chromatograms acquired a sugar, a sugar to the internal standard with respect to the relative intensity is calculated by, the sugar chain binding site to a sugar chain relative quantitation, including quantitative sugar chain, this analysis unit, and outputs the result of the analysis program for causing a computer functions as the output unit.
[claim11]
11. The amino acid sequence of a peptide EEQYNSTYR,EEQFNSTFR,EEQYNSTFR, EEQFNSTYR or asparagine residue, GlcNAc residues 1, 1 or 1 of the residues GlcNAc residues Fuc is bonded is formed by connecting, IgG antibody-of a sugar included trypsin digestion peptide for quantitative analysis of internal standard peptides.
[claim12]
12. Type IgG antibody fragments the peptide mixture separated and collected from a sugar, a sugar acetylglucosaminidase and end - β-N - after a reaction, a sugar chain is a non-cut position of the sugar theevaporation peptide, sugar chain of the antibody-IgG for quantitative analysis of the manufacturing method of the internal standard.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • YOKOHAMA CITY UNIVERSITY
  • 発明者(英語)
  • OHTA YUKI
  • KAWASAKI NANA
  • TAKAKURA DAISUKE
国際特許分類(IPC)
指定国 (WO201834346)
National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN BR BW BY BZ CA CH CL CN CO CR CU CZ DE DJ DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IR IS JO JP KE KG KH KN KP KR KW KZ LA LC LK LR LS LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PA PE PG PH PL PT QA RO RS RU RW SA SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW KM ML MR NE SN ST TD TG
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