Development of a method to analyze glycosylation site-specific glycome at proteome-scale for study on significance of glycan heterogeneity

• Project/Area Number: 15K14426
• Research Category: Grant-in-Aid for Challenging Exploratory Research
• Allocation Type: Multi-year Fund
• Research Field: Genome biology
• Research Institution: National Institute of Advanced Industrial Science and Technology
• Principal Investigator: Kaji Hiroyuki 国立研究開発法人産業技術総合研究所, 創薬基盤研究部門, 研究グループ長 (80214302)
• Research Collaborator: TOMIOKA Azusa ; FUJITA Mika ; SUKEGAWA Masako ; OKATANI Chiaki ; SHIKANAI Toshihide ; NARIMATSU Hisashi
• Project Period (FY): 2015-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥3,900,000 (Direct Cost: ¥3,000,000、Indirect Cost: ¥900,000); Fiscal Year 2016: ¥1,690,000 (Direct Cost: ¥1,300,000、Indirect Cost: ¥390,000); Fiscal Year 2015: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: グライコプロテオミクス / 糖鎖修飾 / 翻訳後修飾 / 質量分析 / 糖鎖不均一性 / マウス / グライコーム / プロテオミクス / グリコシレーション

Outline of Final Research Achievements

Since glycans on glycoproteins are synthesized by many glycan synthesis-related enzymes, they have diverse and heterogeneous structures. Since the expression profile of the enzyme varies depending on the cell type, the glycan variety (glycomes) varies from cell to cell. Furthermore, glycomes are recognized to be different for each type of protein, and at each attachment site. However, little is known about the actual state of glycan heterogeneity. Direct analysis of glycopeptides is essential for analyzing site-specific glycomes, and several methods using MS analysis based on multi-step dissociation have been developed. However, its sensitivity is quite low. Therefore, we developed an MS/MS-independent method for site-specific glycome analysis, and applied it to the glycopeptides obtained from several major mouse tissues. We could obtain large scale information of site specific glycomes; about 12,000 site-specific glycoforms at about 1,200 sites on ca 800 glycoproteins.

Research Products

• [Journal Article] Efficient production of glycosylated Cypridina luciferase using plant cells. (2017)
  • Author(s): Mitani Y, Oshima Y, Mitsuda N, Tomioka A, Sukegawa M, Fujita M, Kaji H, Ohmiya Y.
  • Journal Title: Protein Expr Purif. Volume: 133 Pages: 102-109
  • DOI: 10.1016/j.pep.2017.03.008
  • Peer Reviewed
• [Presentation] 糖タンパク質大規模解析とデータベース構築： 糖鎖付加位置マッピングから位置ごとのグライコーム解析へ (2016)
  • Author(s): 梶 裕之, 富岡あづさ, 野呂絵里花, 藤田弥佳, 助川昌子, 岡谷千晶, 鹿内俊秀, 成松久
  • Organizer: 日本分子生物学会年会
  • Place of Presentation: パシフィコ横浜（神奈川県横浜市）
  • Year and Date: 2016-11-30
  • Invited
• [Presentation] 最近のグライコプロテオミクス： 糖鎖付加部位ごとの糖鎖不均一性の大規模解析法の開発 (2016)
  • Author(s): 梶 裕之
  • Organizer: 日本糖鎖科学コンソーシアムシンポジウム
  • Place of Presentation: ソラシティカンファレンスセンター（東京都千代田区）
  • Year and Date: 2016-11-01
  • Invited
• [Presentation] 糖タンパク質の糖鎖不均一性の大規模解析 (2016)
  • Author(s): 梶 裕之, 富岡あづさ, 野呂絵里花, 藤田弥佳, 助川昌子, 岡谷千晶, 鹿内俊秀, 成松久
  • Organizer: 日本生化学会大会
  • Place of Presentation: 仙台国際センター（宮城県仙台市）
  • Year and Date: 2016-09-27
  • Invited
• [Presentation] Accurate mass- and glycan composition-based assignment of glycosylation site-specific glycomes of complex glycoprotein mixture (2016)
  • Author(s): 梶 裕之, 富岡あづさ, 野呂絵里花, 藤田弥佳, 助川昌子, 岡谷千晶, 鹿内俊秀, 成松久
  • Organizer: Human Proteome Organization World Congress
  • Place of Presentation: Taipei International Convention Center（台北、台湾）
  • Year and Date: 2016-09-20
  • Int'l Joint Research / Invited
• [Presentation] グライコプロテオミクス： 糖鎖付加位置特異的グライコームの大規模分析 (2016)
  • Author(s): 梶 裕之, 富岡あづさ, 野呂絵里花, 藤田弥佳, 助川昌子, 岡谷千晶, 鹿内俊秀, 成松久
  • Organizer: 日本プロテオーム学会2016年大会
  • Place of Presentation: 北里大学薬学部（東京都港区）
  • Year and Date: 2016-07-28
• [Presentation] 糖鎖付加部位ごとのグライコーム分析法の開発と大規模分析への応用 (2015)
  • Author(s): 梶 裕之、富岡 あづさ、野呂 絵里花、 鹿内 俊秀、成松 久
  • Organizer: BMB2015（日本分子生物学会、日本生化学会合同大会）
  • Place of Presentation: 神戸国際会議場、兵庫県
  • Year and Date: 2015-12-01
  • Invited
• [Presentation] LC/MS-based large-scale analysis of site-specific heterogeneity of mouse glycoproteins (2015)
  • Author(s): Hiroyuki Kaji, Azusa Tomioka, Mika Fujita, Masako Sukegawa, Erika Noro, Chiaki Okatani, Toshihide Shikanai, & Hisashi Narimatsu
  • Organizer: 7th ACGG (Asian Community for Glycoscience and Glycotechnology)
  • Place of Presentation: 宮城県 松島町
  • Year and Date: 2015-11-14
  • Int'l Joint Research / Invited
• [Presentation] Development of a method for large-scale analysis of the site-specific glycomes of glycoproteins (2015)
  • Author(s): Hiroyuki Kaji, Azusa Tomioka, Erika Noro, Toshihide Shikanai, Hisashi Narimatsu.
  • Organizer: 14th HUPO World Congress
  • Place of Presentation: Vancouver Convention Centre, Canada
  • Year and Date: 2015-09-28
  • Int'l Joint Research
• [Presentation] 糖鎖付加部位ごとの糖鎖不均一性を大規模に解析する方法の開発 (2015)
  • Author(s): 梶 裕之、富岡あづさ、鹿内俊秀、成松 久
  • Organizer: 第34回日本糖質学会年会
  • Place of Presentation: 東京都文京区 東京大学
  • Year and Date: 2015-07-31
• [Presentation] 糖鎖付加部位特異的グライコーム大規模分析法の開発と応用 (2015)
  • Author(s): 梶 裕之、富岡あづさ、野呂絵里花、藤田弥佳、成松 久
  • Organizer: 日本プロテオーム学会2015年会
  • Place of Presentation: 熊本市 熊本森都心プラザ
  • Year and Date: 2015-07-23
• [Book] 糖鎖を狙う (2017)
  • Author(s): 梶 裕之、富岡あづさ、野呂絵里花、栂谷内晶、佐藤隆
  • Total Pages: 7
  • Publisher: 羊土社
• [Remarks] GlycoProtDB: A glycoprotein database
  • URL: http://acgg.asia/gpdb2/index?request_locale=ja