Characterization of Membrane and Cytosolic Proteins of Erythrocytes

  • Gloria Alvarez-Llamas
  • Fernando de la Cuesta
  • Maria G. Barderas
  • Irene Zubiri
  • Maria Posada-Ayala
  • Fernando Vivanco
Part of the Methods in Molecular Biology book series (MIMB, volume 1000)


With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, this chapter details a novel approach that allows the analysis of both human cytosolic and membrane sub-proteomes. Despite their simple structure, the high content of hemoglobin present in the red blood cells (RBCs) makes their proteome analysis enormously difficult. Careful investigation of different strategies for isolation of the membrane and cytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE was carried out, paying particular attention to hemoglobin removal. As result, a simple, quick, and satisfactory approach for hemoglobin depletion of erythrocyte cells based on HemogloBind™ reagent is shown here to satisfactorily analyze the cytosolic sub-proteome by 2-DE without major interference. For membrane proteome, a novel combined strategy based on hypotonic lysis isolation and further purification on minicolumns is described, allowing detection of high-molecular-weight proteins (i.e., spectrin, ankyrin) and well-resolved 2-DE patterns. The analysis of the membrane fraction by nano-LC coupled to an LTQ-Orbitrap mass spectrometer results in the identification of a total of 188 unique proteins.

Key words

Erythrocytes Hemoglobind Membrane proteins Cytosolic proteins 



This work was supported by Instituto de Salud Carlos III (grant numbers FIS PI070537, FIS PI080970, FISPI080920, RD07/0064/0023, FI06/00583, CP09/00229, PI11/02239, PI11/001401), Mutua Madrileña Automovilista (grant number 20174/004), Fundacion Conchita Rabago, Redes temáticas de Investigación Cooperativa en Salud (RD06/0014/1015), and grants from Fundación para la Investigación Sanitaria de Castilla-La Mancha (FISCAM PI2008-08, PI2008-28, PI2008-52).


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Copyright information

© Springer Science+Business Media New York 2013

Authors and Affiliations

  • Gloria Alvarez-Llamas
    • 1
  • Fernando de la Cuesta
    • 2
  • Maria G. Barderas
    • 3
  • Irene Zubiri
    • 4
  • Maria Posada-Ayala
    • 4
  • Fernando Vivanco
    • 1
    • 5
  1. 1.Department of ImmunologyIIS-Fundacion Jimenez DiazMadridSpain
  2. 2.Department of Vascular PhysiopathologyHospital Nacional de Parapléjicos, SESCAMToledoSpain
  3. 3.Department of Vascular Physiopathology, SESCAMHospital Nacional de ParapléjicosToledoSpain
  4. 4.Department of ImmunologyInstituto de Investigacion Sanitaria Fundacion Jimenez Diaz (IIS-FJD)MadridSpain
  5. 5.Department of Biochemistry and Molecular Biology IUniversidad ComplutenseMadridSpain

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