A novel methodology for the analysis of membrane and cytosolic sub-proteomes of erythrocytes by 2-DE
- Darde, Veronica M. 3
- Vivanco, Fernando 15
- Barderas, Maria G. 2
- Caramelo, Carlos 4
- Zubiri, Irene 1
- Alvarez-Llamas, Gloria 1
- de la Cuesta, Fernando 1
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1
Fundación Jiménez Díaz
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- 2 Department of Vascular Physiopathology, Hospital Nacional de Parapléjicos, SESCAM
- 3 Proteomics Unit, Hospital Nacional de Parapléjicos, SESCAM
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4
Universidad Autónoma de Madrid
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5
Universidad Complutense de Madrid
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ISSN: 0173-0835, 1522-2683
Year of publication: 2009
Volume: 30
Issue: 23
Pages: 4095-4108
Type: Article
More publications in: ELECTROPHORESIS
Abstract
With the aim of studying a wide cohort of erythrocyte samples in a clinical setting, wepropose here a novel approach that allows the analysis of both human cytosolic andmembrane sub-proteomes. Despite their simple structure, the high content of hemo-globin present in the red blood cells (RBCs) makes their proteome analysis enormouslydifficult. We investigate here different strategies for isolation of the membrane andcytosolic fractions from erythrocytes and their influence on proteome profiling by 2-DE,paying particular attention to hemoglobin removal. A simple, quick and satisfactoryapproach for hemoglobin depletion based on HemogloBindTMreagent was satisfactorilyapplied to erythrocyte cells, allowing the analysis of the cytosolic sub-proteome by 2-DEwithout major interference. For membrane proteome, a novel combined strategy basedon hypotonic lysis isolation and further purification on minicolumns is described here,allowing detection of high molecular weight proteins (i.e. spectrin, ankyrin) and well-resolved 2-DE patterns. An aliquot of the membrane fraction was also in solutiondigested and analyzed by nano-LC coupled to an LTQ-Orbitrap mass spectrometer. Atotal of 188 unique proteins were identified by this approach. This study sets the basis forfuture clinical studies where the erythrocyte cell may be implicated