title: Characterization of membrane and cytosolic proteins of erythrocytes.
abstract: 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.
authors.affiliation: Department of Immunology, IIS-Fundacion Jimenez Diaz, Madrid, Spain.
authors.forename: Gloria
authors.initials: G
authors.lastname: Alvarez-Llamas
authors.forename: Fernando
authors.initials: F
authors.lastname: de la Cuesta
authors.forename: Maria G
authors.initials: MG
authors.lastname: Barderas
authors.forename: Irene
authors.initials: I
authors.lastname: Zubiri
authors.forename: Maria
authors.initials: M
authors.lastname: Posada-Ayala
authors.forename: Fernando
authors.initials: F
authors.lastname: Vivanco
mesh_terms: D001800:Blood Specimen Collection; D002469:Cell Separation; D002853:Chromatography, Liquid; D003600:Cytosol / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000166:cytology*; D006454:Hemoglobins / Q000302:isolation & purification; D006801:Humans; D008565:Membrane Proteins / Q000302:isolation & purification / Q000378:metabolism*; D036103:Nanotechnology; D040901:Proteomics / Q000379:methods*; D053719:Tandem Mass Spectrometry; D013997:Time Factors
chemical_list: D006454:Hemoglobins; D008565:Membrane Proteins