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Molecular & Cellular Proteomics 7:2254-2269, 2008.
© 2008 by The American Society for Biochemistry and Molecular Biology, Inc.

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Research

Extensive Analysis of the Cytoplasmic Proteome of Human Erythrocytes Using the Peptide Ligand Library Technology and Advanced Mass Spectrometry^*,S

Florence Roux-Dalvai^,, Anne Gonzalez de Peredo^,, Carolina Simó^¶, Luc Guerrier^||, David Bouyssié, Alberto Zanella^**, Attilio Citterio^¶, Odile Burlet-Schiltz, Egisto Boschetti^||, Pier Giorgio Righetti^¶, and Bernard Monsarrat^,

From the Institute of Pharmacology and Structural Biology, CNRS, Université de Toulouse, 31077 Toulouse, France, ^|| Bio-Rad C/o Commissariat à l'Energie Atomique-Saclay, 91191 Gif-sur-Yvette, France, ^** Division of Hematology, Fondazione IRCCS Ospedale Maggiore Policlinico, Mangiagalli e Regina Elena, Milan, Italy, and ^¶ Polytechnic of Milan, Milan 20133, Italy

The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored. Here we used a combinatorial library of hexameric peptides as a capturing agent to lower the signal of hemoglobin and amplify the signal of low to very low abundance proteins in the cytoplasm of human red blood cells (RBCs). Two types of hexapeptide library beads have been adopted: amino-terminal hexapeptide beads and beads in which the peptides have been further derivatized by carboxylation. The amplification of the signal of low abundance and suppression of the signal of high abundance species were fully demonstrated by two-dimensional gel maps and nano-LC-MSMS analysis. The effect of this new methodology on quantitative information also was explored. Moreover using this approach on an LTQ-Orbitrap mass spectrometer, we could identify with high confidence as many as 1578 proteins in the cytoplasmic fraction of a highly purified preparation of RBCs, allowing a deep exploration of the classical RBC pathways as well as the identification of unexpected minor proteins. In addition, we were able to detect the presence of eight different hemoglobin chains including embryonic and newly discovered globin chains. Thus, this extensive study provides a huge data set of proteins that are present in the RBC cytoplasm that may help to better understand the biology of this simplified cell and may open the way to further studies on blood pathologies using targeted approaches.

Supported by the CNRS, the Génopole Toulouse Midi-Pyrénées, the Région Midi-Pyrénées, and grants from the Institut National du Cancer, Agence Nationale de la Recherche, and Fondation pour la Recherche Médicale. To whom correspondence may be addressed: Inst. de Pharmacologie et de Biologie Structurale, 205 route de Narbonne, 31077 Toulouse cedex 4, France. E-mail: bernard.monsarrat{at}ipbs.fr

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