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Analysis of Protein Glycation Using Phenylboronate Acrylamide Gel Electrophoresis

  • Marta P. Pereira Morais
  • John S. Fossey
  • Tony D. James
  • Jean M. H. van den Elsen
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 869)

Abstract

Carbohydrate modification of proteins adds complexity and diversity to the proteome. However, undesired carbohydrate modifications also occur in the form of glycation, resulting in diseases such as diabetes, Alzheimer’s disease, autoimmune diseases, and cancer. The analysis of glycated proteins is challenging due to their complexity and variability. Numerous analytical techniques have been developed that require expensive specialised equipment and complex data analysis. In this chapter, we describe a simple electrophoresis-based method that enables users to detect, identify, and analyze these post-translational modifications. This new cost-effective methodology will aid the detection of unwanted glycation products in processed foods and may lead to new diagnostics and therapeutics for age-related chronic diseases and glycosylation disorders.

Key words

Protein glycation Gel electrophoresis Boronic acid Step gel 2D gel 

Notes

Acknowledgements

This work was supported by the Biotechnology and Biological Sciences Research Council (BBSRC) (MPPM). We are grateful for the University of Bath Research Development & Support Office (RDSO) for funding.

References

  1. 1.
    Ulrich P, Cerami A (2001) Protein glycation, diabetes, and aging. Recent Prog Horm Res 56:1–22PubMedCrossRefGoogle Scholar
  2. 2.
    Lapolla A, Traldi P, Fedele D (2005) Importance of measuring products of non-enzymatic glycation of proteins. Clin Biochem 38:103–115PubMedCrossRefGoogle Scholar
  3. 3.
    Pokharna H, Pottenger L (1997) Nonenzymatic glycation of cartilage proteoglycans: an in vivo and in vitro study. Glycoconj J 14:917–923PubMedCrossRefGoogle Scholar
  4. 4.
    Geoghegan KF, Dixon HBF, Rosner PJ et al (1999) Spontaneous (alpha)-N-6-phosphogluconoylation of a “His Tag” in Escherichia coli: the cause of extra mass of 258 or 178 Da in fusion proteins. Anal Biochem 267:169–184PubMedCrossRefGoogle Scholar
  5. 5.
    Yan Z, Caldwell GW, McDonell PA (1999) Identification of a gluconic acid derivative attached to the N-terminus of histidine-tagged proteins expressed in bacteria. Biochem Biophys Res Commun 262:793–800PubMedCrossRefGoogle Scholar
  6. 6.
    Monnier VM, Cerami A (1981) Nonenzymatic browning in vivo: possible process for aging of long-lived proteins. Science 211:491–493PubMedCrossRefGoogle Scholar
  7. 7.
    Foerster A, Henle T (2003) Glycation in food and metabolic transit of dietary AGEs (advanced glycation end-products): studies on the urinary excretion of pyrraline. Biochem Soc Trans 31:1383–1385PubMedCrossRefGoogle Scholar
  8. 8.
    Montgomery H, Tanaka K, Belgacem O (2010) Glycation pattern of peptides condensed with maltose, lactose and glucose determined by ultraviolet matrix-assisted laser desorption/ionization tandem mass spectrometry. Rapid Commun Mass Spectrom 24: 841–848PubMedCrossRefGoogle Scholar
  9. 9.
    Klenk DC, Hermanson GT, Krohn RI et al (1982) Determination of glycosylated hemoglobin by affinity chromatography: comparison with colorimetric and ion-exchange methods, and effects of common interferences. Clin Chem 28:2088–2094PubMedGoogle Scholar
  10. 10.
    Zhang Q, Ames JM, Smith RD et al (2008) A perspective on the maillard reaction and the analysis of protein glycation by mass spectrometry: probing the pathogenesis of chronic disease. J Proteome Res 8:754–769CrossRefGoogle Scholar
  11. 11.
    Wu JT, Tu M-C, Zhung P (1996) Advanced glycation end product (AGE): characterization of the products from the reaction between D-glucose and serum albumin. J Clin Lab Anal 10:21–34PubMedCrossRefGoogle Scholar
  12. 12.
    Weber K, Osborn M (1969) The reliability of molecular weight determinations by dodecyl sulfate-polyacrylamide gel electrophoresis. J Biol Chem 244:4406–4412PubMedGoogle Scholar
  13. 13.
    Pereira Morais MP, Mackay JD, Bhamra SK et al (2009) Analysis of protein glycation using phenylboronate acrylamide gel electrophoresis. Proteomics 10:48–58CrossRefGoogle Scholar
  14. 14.
    Davis BJ, Ornstein L (1959) A new high resolution electrophoresis method. In: Society for the study of blood, New York Academy of MedicineGoogle Scholar
  15. 15.
    Raymond S, Weintraub L (1959) Acrylamide gel as a supporting medium for zone electrophoresis. Science 130:711PubMedCrossRefGoogle Scholar
  16. 16.
    Miksík I, Deyl Z (1997) Post-translational non-enzymatic modification of proteins. II. Separation of selected protein species after glycation and other carbonyl-mediated modifications. J Chromatogr B Biomed Sci Appl 699:311–345PubMedCrossRefGoogle Scholar
  17. 17.
    Garfin DE (2003) Gel electrophoresis of proteins. In: Davey J, Lord MJ (eds) Essential cell biology: a practical approach. Oxford University Press, OxfordGoogle Scholar
  18. 18.
    Switzer RC, Merril CR, Shifrin S (1979) A highly sensitive silver stain for detecting proteins and peptides in polyacrylamide gels. Anal Biochem 98:231–237PubMedCrossRefGoogle Scholar
  19. 19.
    Springsteen G, Wang B (2002) A detailed examination of boronic acid-diol complexation. Tetrahedron 58:5291–5300CrossRefGoogle Scholar
  20. 20.
    Lorand JP, Edwards JO (1959) Polyol complexes and structure of the benzeneboronate ion. J Org Chem 24:769–774CrossRefGoogle Scholar
  21. 21.
    Nishiyabu R, Kubo Y, James TD et al (2010) Boronic acid building blocks: tools for sensing and separation. Chem Commun 47: 1106–1123Google Scholar
  22. 22.
    Weith HL, Wiebers JL, Gilham PT (1970) Synthesis of cellulose derivatives containing the dihydroxyboryl group and a study of their capacity to form specific complexes with sugars and nucleic acid components. Biochemistry 9:4396–4401PubMedCrossRefGoogle Scholar
  23. 23.
    Liu X-C (2006) Boronic acids as ligands for affinity chromatography. Chinese J Chromatogr 24:73–80CrossRefGoogle Scholar
  24. 24.
    Cartwright SJ, Waley SG (1984) Purification of beta-lactamases by affinity chromatography on phenylboronic acid-agarose. Biochem J 221: 505–512PubMedGoogle Scholar
  25. 25.
    Armbruster DA (1987) Fructosamine: structure, analysis, and clinical usefulness. Clin Chem 33:2153–2163PubMedGoogle Scholar
  26. 26.
    Priego Capote F, Sanchez J-C (2009) Strategies for proteomic analysis of non-enzymatically glycated proteins. Mass Spectrom Rev 28:135–146PubMedCrossRefGoogle Scholar
  27. 27.
    Jackson TR, Springall JS, Rogalle D et al (2008) Boronate affinity saccharide electrophoresis: a novel carbohydrate analysis tool. Electrophoresis 29:4185–4191PubMedCrossRefGoogle Scholar
  28. 28.
    Jackson P (1990) The use of polyacrylamide-gel electrophoresis for the high-resolution separation of reducing saccharides labelled with the fluorophore 8-aminonaphthalene-1,3,6-trisulphonic acid. Detection of picomolar quantities by an imaging system based on a cooled charge-coupled device. Biochem J 270:705–713PubMedGoogle Scholar
  29. 29.
    Mahoney DJ, Aplin RT, Calabro A et al (2001) Novel methods for the preparation and characterization of hyaluronan oligosaccharides of defined length. Glycobiology 11:1025–1033PubMedCrossRefGoogle Scholar
  30. 30.
    Calabro A, Benavides M, Tammi M et al (2000) Microanalysis of enzyme digests of hyaluronan and chondroitin/dermatan sulfate by fluorophore-assisted carbohydrate electrophoresis (FACE). Glycobiology 10:273–281PubMedCrossRefGoogle Scholar
  31. 31.
    Ma WMJ, Pereira Morais MP, D’Hooge F et al (2009) Dye displacement assay for saccharide detection with boronate hydrogels. Chem Commun 5:532–534Google Scholar
  32. 32.
    Curry S, Mandelkow H, Brick P et al (1998) Crystal structure of human serum albumin complexed with fatty acid reveals an asymmetric distribution of binding sites. Nat Struct Mol Biol 5:827–835CrossRefGoogle Scholar
  33. 33.
    Sattarahmady N, Moosavi-Movahedi AA, Ahmad F et al (2007) Formation of the molten globule-like state during prolonged glycation of human serum albumin. Biochim Biophys Acta 1770:933–942PubMedCrossRefGoogle Scholar
  34. 34.
    Inaba M, Okuno S, Kumeda Y et al, Osaka CKD Expert Research Group (2007) Glycated albumin is a better glycemic indicator than glycated hemoglobin values in hemodialysis patients with diabetes: effect of anemia and erythropoietin injection. J Am Soc Nephrol 18:896–903Google Scholar
  35. 35.
    Burman JD, Leung E, Atkins KL et al (2008) Interaction of human complement with Sbi, a staphylococcal immunoglobulin-binding protein: indications of a novel mechanism of ­complement evasion by Staphylococcus aureus. J Biol Chem 283:17579–17593PubMedCrossRefGoogle Scholar
  36. 36.
    Margolis J, Kenrick KG (1967) Polyacrylamide gel-electrophoresis across a molecular sieve gradient. Nature 214:1334–1336PubMedCrossRefGoogle Scholar
  37. 37.
    O’Farrell PH (1975) High resolution two-dimensional electrophoresis of proteins. J Biol Chem 250:4007–4021PubMedGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2012

Authors and Affiliations

  • Marta P. Pereira Morais
    • 1
  • John S. Fossey
    • 2
  • Tony D. James
    • 3
  • Jean M. H. van den Elsen
    • 1
  1. 1.Department of Biology and BiochemistryUniversity of BathBathUK
  2. 2.School of Chemistry, University of BirminghamEdgbastonUK
  3. 3.Department of ChemistryUniversity of BathBathUK

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