title: Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry.
abstract: 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.
authors.affiliation: Institute of Pharmacology and Structural Biology, CNRS, Université de Toulouse, 31077 Toulouse, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Roux-Dalvai
authors.forename: Anne
authors.initials: A
authors.lastname: Gonzalez de Peredo
authors.forename: Carolina
authors.initials: C
authors.lastname: Simó
authors.forename: Luc
authors.initials: L
authors.lastname: Guerrier
authors.forename: David
authors.initials: D
authors.lastname: Bouyssié
authors.forename: Alberto
authors.initials: A
authors.lastname: Zanella
authors.forename: Attilio
authors.initials: A
authors.lastname: Citterio
authors.forename: Odile
authors.initials: O
authors.lastname: Burlet-Schiltz
authors.forename: Egisto
authors.initials: E
authors.lastname: Boschetti
authors.forename: Pier Giorgio
authors.initials: PG
authors.lastname: Righetti
authors.forename: Bernard
authors.initials: B
authors.lastname: Monsarrat
mesh_terms: D001798:Blood Proteins / Q000032:analysis* / Q000302:isolation & purification; D003593:Cytoplasm / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional; D004912:Erythrocytes / Q000737:chemistry*; D006801:Humans; D019151:Peptide Library; D020543:Proteome / Q000032:analysis / Q000302:isolation & purification; D040901:Proteomics; D053719:Tandem Mass Spectrometry / Q000379:methods
chemical_list: D001798:Blood Proteins; D019151:Peptide Library; D020543:Proteome