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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.
従来技術、競合技術の概要(英語) BACKGROUND ART
Sugar chain is an important posttranslational modifications of proteins and, in recent years, analysis of their structure and function has been promoted rapidly. Such glycoproteins, glycopeptides as a method of analyzing, liquid chromatography/mass spectrometry method using the adapted to be widely used. Liquid chromatography/mass spectrometry such as N-linked glycoproteins used, when analyzing the structure of a peptide, a sugar chain of the peptide were deglycosylated -N- (PNGase F) by the cutting is performed, N-linked sugar chain bound to asparagine (Asn) aspartic acid (Asp) is changed to, by detecting the change in N-linked sugar chains generally the method of determining the position. (For example Patent Document 1, Non-Patent Document 1 reference).
However, N-linked sugar chain binding using PNGase F is located at a position to observe the change of the Asn Asp to a method, according to the Asp Asn deamidation or a change, due to the variation of the Asp PNGase F processing cannot determine whether the problem, the change in mass of from +1 Da to Asn Asp small as, erroneous identification of one problem tends to frequently 2.
As a measure for these problems, for example, stable isotope labeled H218 Oin the PNGase F sugar chain can be cut, sugar chain binding position of the Asn to Asp to change when the hydroxy groups of the carboxyl groups of Asp18 Olabeled with, +3 Da the weight change can be differentiated and deamidation of Asn, mono isotopic peak picking of the method for mitigating the identified error due to the used (for example see Non-Patent Document 1).
Glycoproteins, glycopeptides released from of the sugar chain is in relation to, N-linked sugar chains PNGase F all of the free and disconnected from a protein, sugar chain is labeled with a liquid chromatography and the like may be measured using a known method (for example see Patent Document 2).
  • 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 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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