Characterization of Membrane and Cytosolic Proteins of Erythrocytes
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 wordsErythrocytes 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).
- 8.Kakhniashvili DG, Griko NB, Bulla LA Jr, Goodman SR (2005) The proteomics of sickle cell disease: profiling of erythrocyte membrane proteins by 2D-DIGE and tandem mass spectrometry. Exp Biol Med 230:787–792Google Scholar