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Discrimination of Glycoproteins from Unglycosylated Proteins in Capillary Electrophoresis: Two-Color LIF Detection Coupled with Post-column Derivatization

  • Takashi Kaneta
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1466)

Abstract

Glycosylation is one of the most important posttranslational modifications (PTMs) which lead to the functionalization of proteins. Here, we describe one method for discriminating glycosylated proteins from unglycosylated ones in their mixture sample by capillary electrophoretic separation and two-color laser-induced fluorescence detection coupled with post-column derivatization. Two lasers emitting at 450 and 532 nm permit the detection of amino groups of proteins derivatized by naphthalene-2,3-dicarboxaldehyde and a fluorescently labeled lectin, tetramethylrhodamine-labeled concanavalin A (Rh-Con A), respectively. When a protein mixture react with Rh-Con A, the glycoproteins bound with Rh-Con A exhibit signals at the same migration time in two electropherograms obtained by 450- and 532-nm lasers whereas unbound proteins show a signal only in the electropherogram of the 450-nm laser. So, when one protein is glycosylated it is detected at the same migration time in the electropherograms obtained by two lasers.

Key words

Capillary electrophoresis Glycoprotein Postcolumn derivatization Two-color laser-induced fluorescence 

Notes

Acknowledgement

This research was supported by Grants-in-Aid for Scientific Research, Grant-in-Aid for challenging Exploratory Research (No. 25620114) and Scientific Research (B) (No. 26288067).

References

  1. 1.
    Tian Y, Zhang H (2010) Glycoproteomics and clinical applications. Proteomics Clin Appl 4:124–132CrossRefPubMedGoogle Scholar
  2. 2.
    Carr SA, Huddleston MJ, Bean MF (1993) Selective identification and differentiation of N- and O-linked oligosaccharides in glycoproteins by liquid chromatography–mass spectrometry. Protein Sci 2:183–196CrossRefPubMedPubMedCentralGoogle Scholar
  3. 3.
    Balaguer E, Neusüss C (2006) Glycoprotein characterization combining intact protein and glycan analysis by capillary electrophoresis–electrospray ionization-mass spectrometry. Anal Chem 78:5384–5393CrossRefPubMedGoogle Scholar
  4. 4.
    Balaguer E, Demelbauer U, Pelzing M, Sanz-Nebot V, Barbosa J, Neusüß C (2006) Glycoform characterization of erythropoietin combining glycan and intact protein analysis by capillary electrophoresis–electrospray–time-of-flight mass spectrometry. Electrophoresis 27:2638–2650CrossRefPubMedGoogle Scholar
  5. 5.
    Nakano M, Higo D, Arai E, Nakagawa T, Kakehi K, Taniguchi N, Kondo A (2009) Capillary electrophoresis–electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives. Glycobiology 19:135–143CrossRefPubMedGoogle Scholar
  6. 6.
    Lamari F, Militsopoulou M, Gioldassi X, Karamanos NK (2001) Capillary electrophoresis: a superior miniaturized tool for analysis of the mono-, di-, and oligosaccharide constituents of glycan moieties in proteoglycans. Fresenius J Anal Chem 371:157–167CrossRefPubMedGoogle Scholar
  7. 7.
    Kakehi K, Kinoshita M, Kawakami D, Tanaka J, Sei K, Endo K, Oda Y, Iwaki M, Masuko T (2001) Capillary electrophoresis of sialic acid-containing glycoprotein. Effect of the heterogeneity of carbohydrate chains on glycoform separation using an α1-acid glycoprotein as a model. Anal Chem 73:2640–2647CrossRefPubMedGoogle Scholar
  8. 8.
    Werner WE, Demorest DM, Wiktorowicz JE (1993) Automated Ferguson analysis of glycoproteins by capillary electrophoresis using a replaceable sieving matrix. Electrophoresis 14:759–763CrossRefPubMedGoogle Scholar
  9. 9.
    Weiss S (1999) Fluorescence spectroscopy of single biomolecules. Science 283:1676–1683CrossRefPubMedGoogle Scholar
  10. 10.
    Zhang ZR, Krylov S, Arriaga EA, Polakowski R, Dovichi NJ (2000) One-dimensional protein analysis of an HT29 human colon adenocarcinoma cell. Anal Chem 72:318–322CrossRefPubMedGoogle Scholar
  11. 11.
    Gilman SD, Pietron JJ, Ewing AG (1994) Post-column derivatization in narrow-bore capillaries for the analysis of amino acids and proteins by capillary electrophoresis with fluorescence detection. J Microcol Sep 6:373–384CrossRefGoogle Scholar
  12. 12.
    Gilman SD, Ewing AG (1995) Analysis of single cells by capillary electrophoresis with on-column derivatization and laser-induced fluorescence detection. Anal Chem 67:58–64CrossRefPubMedGoogle Scholar
  13. 13.
    Kaneta T, Yamamoto D, Imasaka T (2009) Postcolumn derivatization of proteins in capillary sieving electrophoresis/laser-induced fluorescence detection. Electrophoresis 30:3780–3785CrossRefPubMedGoogle Scholar
  14. 14.
    Kaneta T, Ogura T, Imasaka T (2011) Analysis of proteins in biological samples by capillary sieving electrophoresis with postcolumn derivatization/laser-induced fluorescence detection. Electrophoresis 32:1061–1067CrossRefPubMedGoogle Scholar
  15. 15.
    Tabara A, Kaneta T (2013) Discrimination of glycoproteins via two-color laser-induced fluorescence detection coupled with postcolumn derivatization in capillary electrophoresis. Electrophoresis 34:2316–2322CrossRefPubMedGoogle Scholar
  16. 16.
    Rose DJ Jr, Jorgenson JW (1988) Post-capillary fluorescence detection in capillary zone electrophoresis using o-phthaldialdehyde. J Chromatogr 447:117–131CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Department of Chemistry, Graduate School of Natural Science and TechnologyOkayama UniversityOkayama-shiJapan

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