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Subcellular Fractionation of Brain Tissue Using Free-Flow Electrophoresis

  • Markus IslingerEmail author
  • Joachim Kirsch
  • Sabine Angermüller
  • Ramona Rotaru
  • Afsaneh Abdolzade-Bavil
  • Gerhard Weber
Protocol
Part of the Neuromethods book series (NM, volume 57)

Abstract

Accurate annotation of protein identifications in organellar proteomics highly depends on the sample quality with special respect to contaminations from other subcellular compartments. In this respect, Free-flow electrophoresis (FFE) offers a valuable alternative to classical centrifugation techniques, since it relies on quite different physical parameters. During the last years, FFE has been successfully used for the separation of various organelles from different tissues, yet is largely unknown in the field of neurobiology. Here we present two separation schemes for the fractionation of a synaptic preparation from rat brain using different modes of FFE. Isotachophoresis (ITP), a focusing technique separating organelles according to their electrophoretic mobilities, was able to distribute the synaptosome sample into different subfractions: mitochondrial cross contaminations showed the highest electrophoretic mobility and migrated nearest to the anode of the FFE instrument; proximate to these, proteins of the presynaptic compartment accumulated, whereas nearest to the cathode of the instrument postsynaptic marker proteins were predominantly found. As a nonfocusing technique, zonal FFE does not possess a separation capacity comparable to ITP; however, due to a continuous separation mode, it is adapted to process higher sample amounts and can be used for large-scale separations. We applied zonal FFE to the same starting material as in ITP and were able to separate mitochondria from synaptic material of the preparation, thus offering a fast alternative to clean synaptosome preparations from residual mitochondrial contaminations.

Key words

Synaptosomes Mitochondria Organelle separation Electrophoresis Proteomics 

Notes

Acknowledgments

We thank Medea Krapp, Heribert Mohr, Ute Sukopp, and Inge Frommer for their excellent technical assistance. We are grateful to Prof. Alfred Völkl for carefully reading through this manuscript.

References

  1. 1.
    Brunet S, Thibault P, Gagnon E, et al. Organelle proteomics: looking at less to see more. Trends Cell Biol 2003;13:629–38.PubMedCrossRefGoogle Scholar
  2. 2.
    Au CE, Bell AW, Gilchrist A, et al. Organellar proteomics to create the cell map. Curr Opin Cell Biol 2007;19:376–85.PubMedCrossRefGoogle Scholar
  3. 3.
    Tribl F, Meyer HE, Marcus, K. Analysis of organelles within the nervous system: impact on brain and organelle functions. Expert Rev Proteomics 2008;5:333–51.PubMedCrossRefGoogle Scholar
  4. 4.
    Grant SG. The synapse proteome and phosphoproteome: a new paradigm for synapse biology. Biochem Soc Trans 2006;34:59–63.PubMedCrossRefGoogle Scholar
  5. 5.
    Li, KW, Jimenez CR. Synapse proteomics: current status and quantitative applications. Expert Rev Proteomics 2008;5:353–60.PubMedCrossRefGoogle Scholar
  6. 6.
    Venable JD, Wohlschlegel J, McClatchy DB, et al. Relative quantification of stable isotope labeled peptides using a linear ion trap-Orbitrap hybrid mass spectrometer. Anal Chem 2007;79:3056–64.PubMedCrossRefGoogle Scholar
  7. 7.
    Mello CF, Sultana R, Piroddi M, et al. Acrolein induces selective protein carbonylation in synaptosomes. Neuroscience 2007;147:674–9.PubMedCrossRefGoogle Scholar
  8. 8.
    Ghijsen WE, Leenders AG, Lopes da Silva FH. Regulation of vesicle traffic and neurotransmitter release in isolated nerve terminals. Neurochem Res 2003;28:1443–52.PubMedCrossRefGoogle Scholar
  9. 9.
    Bai F, Witzmann FA. Synaptosome proteomics. Subcell Biochem 2007;43:77–98.PubMedCrossRefGoogle Scholar
  10. 10.
    Boyd-Kimball D, Castegna A, Sultana R, et al. Proteomic identification of proteins oxidized by Abeta(1–42) in synaptosomes: implications for Alzheimer’s disease. Brain Res 2005;1044:206–15.PubMedCrossRefGoogle Scholar
  11. 11.
    Gillardon F, Rist W, Kussmaul L, et al. Proteomic and functional alterations in brain mitochondria from Tg2576 mice occur before amyloid plaque deposition. Proteomics 2007;7:605–16.PubMedCrossRefGoogle Scholar
  12. 12.
    Barkla BJ, Vera-Estrella R, Pantoja O. Enhanced separation of membranes during free flow zonal electrophoresis in plants. Anal Chem 2007;79:5181–7.PubMedCrossRefGoogle Scholar
  13. 13.
    Eubel H, Lee CP, Kuo J, et al. Free-flow electrophoresis for purification of plant mitochondria by surface charge. Plant J 2007;52:583–94.PubMedCrossRefGoogle Scholar
  14. 14.
    Marsh M. Endosome and lysosome purification by free-flow electrophoresis. Methods Cell Biol 1989;31:319–34.PubMedCrossRefGoogle Scholar
  15. 15.
    Marsh M, Kern H, Harms E, et al. Co-fractionation of BHK-21 cell endosomes and lysosomes by free-flow electrophoresis. Prog Clin Biol Res 1988;270:21–33.PubMedGoogle Scholar
  16. 16.
    Völkl A, Mohr H, Fahimi HD. Peroxisome subpopulations of the rat liver. Isolation by immune free flow electrophoresis. J Histochem Cytochem 1999;47:1111–8.PubMedCrossRefGoogle Scholar
  17. 17.
    Zischka H, Larochette N, Hoffmann F, et al. Electrophoretic analysis of the mitochondrial outer membrane rupture induced by per­meability transition. Anal Chem 2008;80:5051–8.PubMedCrossRefGoogle Scholar
  18. 18.
    Zischka H, Weber G, Weber PJ, et al. mproved proteome analysis of Saccharomyces cerevisiae mitochondria by free-flow electrophoresis. Proteomics 2003;3:906–16.PubMedCrossRefGoogle Scholar
  19. 19.
    Islinger M, Li KW, Loos M, et al. Peroxisomes from the heavy mitochondrial fraction: isolation by zonal free flow electrophoresis and quantitative mass spectrometrical characterization. J Proteome Res 2010;9:113–24.PubMedCrossRefGoogle Scholar
  20. 20.
    Malmstrom J, Lee H, Nesvizhskii AI, et al. Optimized peptide separation and identification for mass spectrometry based proteomics via free-flow electrophoresis. J Proteome Res 2006;5:2241–9.PubMedCrossRefGoogle Scholar
  21. 21.
    Moritz, RL, Simpson RJ. Liquid-based free-flow electrophoresis-reversed-phase HPLC: a proteomic tool. Nat Methods 2005;2:863–73.PubMedCrossRefGoogle Scholar
  22. 22.
    Völkl A, Mohr H, Weber G, et al. Isolation of rat hepatic peroxisomes by means of immune free flow electrophoresis. Electrophoresis 1997;18:774–80.PubMedCrossRefGoogle Scholar
  23. 23.
    Weber G, Bauer J. Counterbalancing hydrodynamic sample distortion effects increases resolution of free-flow zone electrophoresis. Electrophoresis 1998;19:1104–9.PubMedCrossRefGoogle Scholar
  24. 24.
    Weber G, Bocek P. Stability of continuous flow electrophoresis. Electrophoresis 1998;19:3094–5.PubMedCrossRefGoogle Scholar
  25. 25.
    Weber G, Bocek P. Interval isotachophoresis for purification and isolation of ionogenic species. Electrophoresis 1998;19:3090–3.PubMedCrossRefGoogle Scholar
  26. 26.
    Weber G, Islinger M, Weber P, et al. Efficient separation and analysis of peroxisomal membrane proteins using free-flow isoelectric focusing. Electrophoresis 2004;25:1735–47.PubMedCrossRefGoogle Scholar
  27. 27.
    Cutillas PR, Biber J, Marks J, et al. Proteomic analysis of plasma membrane vesicles isolated from the rat renal cortex. Proteomics 2005;5:101–12.PubMedCrossRefGoogle Scholar
  28. 28.
    Drews O, Wildgruber R, Zong C, et al. Mammalian proteasome subpopulations with distinct molecular compositions and proteolytic activities. Mol Cell Proteomics 2007;6:2021–31.PubMedCrossRefGoogle Scholar
  29. 29.
    Eubel H, Meyer EH, Taylor NL, et al. Novel proteins, putative membrane transporters, and an integrated metabolic network are revealed by quantitative proteomic analysis of Arabidopsis cell culture peroxisomes. Plant Physiol 2008;148:1809–29.PubMedCrossRefGoogle Scholar
  30. 30.
    Huang S, Taylor NL, Narsai R, et al. Experimental analysis of the rice mitochondrial proteome, its biogenesis, and heterogeneity. Plant Physiol 2009;149:719–34.PubMedCrossRefGoogle Scholar
  31. 31.
    Zischka H, Braun RJ, Marantidis EP, et al. Differential analysis of Saccharomyces cerevisiae mitochondria by free flow electrophoresis. Mol Cell Proteomics 2006;5:2185–200.PubMedCrossRefGoogle Scholar
  32. 32.
    Pfeiffer F, Simler R, Grenningloh G, et al. Monoclonal antibodies and peptide mapping reveal structural similarities between the subunits of the glycine receptor of rat spinal cord. Proc Natl Acad Sci USA 1984;81:7224–7.PubMedCrossRefGoogle Scholar
  33. 33.
    Kyhse-Andersen J. Electroblotting of multiple gels: a simple apparatus without buffer tank for rapid transfer of proteins from polyacrylamide to nitrocellulose. J Biochem Biophys Methods 1984;10:203–9.PubMedCrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media, LLC 2011

Authors and Affiliations

  • Markus Islinger
    • 1
    Email author
  • Joachim Kirsch
  • Sabine Angermüller
  • Ramona Rotaru
  • Afsaneh Abdolzade-Bavil
  • Gerhard Weber
  1. 1.Department of Anatomy and Cell BiologyUniversity of HeidelbergHeidelbergGermany

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