Qualitative and quantitative cellular glycomics of glycosphingolipids based on rhodococcal endoglycosylceramidase-assisted glycan cleavage, glycoblotting-assisted sample preparation, and matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry analysis

Naoki Fujitani, Yasuhiro Takegawa, Yohei Ishibashi, Kayo Araki, Jun Ichi Furukawa, Susumu Mitsutake, Yasuyuki Igarashi, Makoto Ito, Yasuro Shinohara

研究成果: ジャーナルへの寄稿記事

24 引用 (Scopus)

抄録

Glycosphingolipids (GSLs) are crucially important components of the cellular membrane, where they comprise microdomains with many critical biological functions. Despite this fact, qualitative and quantitative techniques for the analysis of GSLs still lag behind the needs of researchers. In this study, a reliable procedure for the elucidation of cellular GSL-glycomes was established based on (a) enzymatic glycan cleavage by endoglycosylceramidases derived from Rhodococcus sp. in combination with (b) glycoblotting-assisted sample preparation. The mixture of endoglycosylceramidase I and II was employed to maximize the release of glycan moieties from the major classes of GSLs (i.e. ganglio-, (neo)lacto- and globo-series GSLs). The glycoblotting technique enabled the quantitative detection of GSL-glycans using as few as 2 × 10 5 cells. Thirty-seven different kinds of cellular GSL glycans were successfully observed in 11 kinds of cells, including Chinese hamster ovary cells and their lectin-resistant mutants as well as murine and human embryonic carcinoma cells. Furthermore, in-depth structural clarification in terms of discrimination of isomers was achieved by MALDI-TOF/TOF mass spectrometry analysis and/or linkage-specific glycosidase digestion. These novel analytical techniques were shown to be capable of delineating cell-specific GSL-glycomes. Thus, they are anticipated to have a broad range of applications for the characterization, description, and comparison of various cellular/tissue samples in the fields of drug discovery and regenerative medicine.

元の言語英語
ページ(範囲)41669-41679
ページ数11
ジャーナルJournal of Biological Chemistry
286
発行部数48
DOI
出版物ステータス出版済み - 12 2 2011

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Glycomics
Glycosphingolipids
Ionization
Mass spectrometry
Polysaccharides
Desorption
Mass Spectrometry
Lasers
Cells
Membrane Microdomains
Rhodococcus
Regenerative Medicine
Glycoside Hydrolases
Matrix-Assisted Laser Desorption-Ionization Mass Spectrometry
Drug Discovery
Cricetulus
Lectins
Isomers
Digestion
Ovary

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

これを引用

Qualitative and quantitative cellular glycomics of glycosphingolipids based on rhodococcal endoglycosylceramidase-assisted glycan cleavage, glycoblotting-assisted sample preparation, and matrix-assisted laser desorption ionization tandem time-of-flight mass spectrometry analysis. / Fujitani, Naoki; Takegawa, Yasuhiro; Ishibashi, Yohei; Araki, Kayo; Furukawa, Jun Ichi; Mitsutake, Susumu; Igarashi, Yasuyuki; Ito, Makoto; Shinohara, Yasuro.

:: Journal of Biological Chemistry, 巻 286, 番号 48, 02.12.2011, p. 41669-41679.

研究成果: ジャーナルへの寄稿記事

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abstract = "Glycosphingolipids (GSLs) are crucially important components of the cellular membrane, where they comprise microdomains with many critical biological functions. Despite this fact, qualitative and quantitative techniques for the analysis of GSLs still lag behind the needs of researchers. In this study, a reliable procedure for the elucidation of cellular GSL-glycomes was established based on (a) enzymatic glycan cleavage by endoglycosylceramidases derived from Rhodococcus sp. in combination with (b) glycoblotting-assisted sample preparation. The mixture of endoglycosylceramidase I and II was employed to maximize the release of glycan moieties from the major classes of GSLs (i.e. ganglio-, (neo)lacto- and globo-series GSLs). The glycoblotting technique enabled the quantitative detection of GSL-glycans using as few as 2 × 10 5 cells. Thirty-seven different kinds of cellular GSL glycans were successfully observed in 11 kinds of cells, including Chinese hamster ovary cells and their lectin-resistant mutants as well as murine and human embryonic carcinoma cells. Furthermore, in-depth structural clarification in terms of discrimination of isomers was achieved by MALDI-TOF/TOF mass spectrometry analysis and/or linkage-specific glycosidase digestion. These novel analytical techniques were shown to be capable of delineating cell-specific GSL-glycomes. Thus, they are anticipated to have a broad range of applications for the characterization, description, and comparison of various cellular/tissue samples in the fields of drug discovery and regenerative medicine.",
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AU - Fujitani, Naoki

AU - Takegawa, Yasuhiro

AU - Ishibashi, Yohei

AU - Araki, Kayo

AU - Furukawa, Jun Ichi

AU - Mitsutake, Susumu

AU - Igarashi, Yasuyuki

AU - Ito, Makoto

AU - Shinohara, Yasuro

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