Summary
Heparins are negatively charged polydispersed linear polysaccharides which have the ability to bind a wide range of biomolecules including enzymes, serine protease inhibitors, growth factors, extracellular matrix proteins, DNA modification enzymes and hormone receptors. In this chromatography, heparin is not only an affinity ligand but also an ion exchanger with high charge density and distribution. Heparin chromatography is an adsorption chromatography in which biomolecules can be specifically and reversibly adsorbed by heparins immobilized on an insoluble support. An advantage of this chromatography is that heparin-binding proteins can be conveniently enriched using its concentration effect. This is especially important for separating low abundance proteins for the analysis in two-dimensional electrophoresis (2DE) or other proteomics approaches. Heparin chromatography is a powerful sample-pretreatment technology that has been widely used to fractionate proteins from extracts of prokaryotic organism or eukaryotic cells. As an example, the fractionation of fibroblast growth factors (FGFs) from the extract of mouse brain microvascular endothelial cells (MVEC) is now introduced to demonstrate the procedure of heparin chromatography.
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References
Rocken, C., Ebert, M.P., Roessner, A. (2004) Proteomics in pathology, research and practice. Pathol. Res. Pract. 200, 69–82.
Betgovargez, E., Knudson, V., Simonian, M.H. (2005) Characterization of proteins in the human serum proteome. J. Biomol. Tech. 16, 306–310.
Yuan, X., Desiderio, D.M. (2005) Proteomics analysis of prefractionated human lumbar cerebrospinal fluid. Proteomics 5, 541–550.
Jarrold, B., DeMuth, J., Greis, K., Burt, T., Wang, F. (2005) An effective skeletal muscle prefractionation method to remove abundant structural proteins for optimized two-dimensional gel electrophoresis. Electrophoresis 26, 2269–2278.
Linke, T., Ross, A.C., Harrison, E.H. (2006) Proteomic analysis of rat plasma by two-dimensional liquid chromatography and matrix-assisted laser desorption ionization time-of-flight mass spectrometry. J. Chromatogr. A [Epub ahead of print].
Guerrera, I.C., Predic-Atkinson, J., Kleiner, O., Soskic, V., Godovac-Zimmermann, J. (2005) Enrichment of phosphoproteins for proteomic analysis using immobilized Fe(III)-affinity adsorption chromatography. J. Proteome. Res. 4, 1545–1553.
Farooqui, A.A. (1980) Purification of enzymes by heparin-sepharose affinity chromatography. J. Chromatogr. 184, 335–345.
Fountoulakis, M., Takacs, B., Langen, H. (1998) Two-dimensional map of basic proteins of Haemophilus influenzae. Electrophoresis 19, 761–766.
Karlsson, G., Winge, S. (2004) Separation of latent, prelatent, and native forms of human antithrombin by heparin affinity high-performance liquid chromatography. Protein Expr. Purif. 33, 339–345.
Staby, A., Sand, M.B., Hansen, R.G., Jacobsen, J.H., Andersen, L.A., Gerstenberg, M., et al. (2005) Comparison of chromatographic ion-exchange resins IV. Strong and weak cation-exchange resins and heparin resins. J. Chromatogr. A 1069, 65–77.
Srivastava, P.N., Farooqui, A.A. (1979) Heparin- sepharose affinity chromatography for purification of bull seminal-plasma hyaluronidase. Biochem. J. 183, 531–537.
Iida, T., Kamo, M., Uozumi, N., Inui, T., Imai, K. (2005) Further application of a two-step heparin affinity chromatography method using divalent cations as eluents: purification and identification of membrane-bound heparin binding proteins from the mitochondrial fraction of HL-60 cells. J Chromatogr. B Analyt. Technol. Biomed. Life Sci. 823, 209–212.
Marques, M.A., Espinosa, B.J., Xavier da Silveira, E.K., Pessolani, M.C., Chapeaurouge, A., Perales, J., et al. (2004) Continued proteomic analysis of Mycobacterium leprae subcellular fractions. Proteomics 4, 2942–2953.
Faham, S., Hileman, R.E., Fromm, J.R., Linhardt, R.J., Rees, D.C. (1996) Heparin structure and interactions with basic fibroblast growth factor. Science 271, 1116–1120.
Katoh, Y., Katoh, M. (2005) Comparative genomics on FGF7, FGF10, FGF22 orthologs, and identification of fgf25. Int. J. Mol. Med. 16, 767–770.
Shastry, S., Tyagi, N., Hayden, M.R., Tyagi, S.C. (2004) Proteomic analysis of homocysteine inhibition of microvascular endothelial cell angiogenesis. Cell. Mol. Biol. (Noisy-le-grand) 50, 931–937.
Amersham Bioscience Inc. Introduction of heparin sepharose 6 fast flow.
Audus, K.L., Borchardt, R.T. (1987) Bovine brain microvessel endothelial cell monolayers as a model system for the blood-brain barrier. Ann. N. Y. Acad. Sci. 507, 9–18.
Shastry, S., Tyagi, S.C. (2004) Homocysteine induces metalloproteinase and shedding of beta-1 integrin in microvessel endothelial cells. J. Cell. Biochem. 93, 207–213.
Shen, H., Cheng, G., Fan, H., Zhang, J., Zhang, X., Lu, H., et al. (2006) Expressed proteome analysis of human hepatocellular carcinoma in nude mice (LCI-D20) with high metastasis potential. Proteomics 6, 528–537.
Hauck, S.M., Schoeffmann, S., Deeg, C.A., Gloeckner, C.J., Swiatek-de Lange, M., Ueffing, M. (2005) Proteomic analysis of the porcine interphotoreceptor matrix. Proteomics 5, 3623–3636.
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Xiong, S., Zhang, L., He, QY. (2008). Fractionation of Proteins by Heparin Chromatography. In: Posch, A. (eds) 2D PAGE: Sample Preparation and Fractionation. Methods in Molecular Biology™, vol 424. Humana Press. https://doi.org/10.1007/978-1-60327-064-9_18
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DOI: https://doi.org/10.1007/978-1-60327-064-9_18
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