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

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. 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. Mature red blood cells (RBCs) 1The abbreviations used are: RBC, red blood cell; WBC, white blood cell; ADH, alcohol dehydrogenase; TUC, 2 m thiourea, 7 m urea, 2% CHAPS; UCA, 9 m urea, citric acid to pH 3.3; HOS, hydro-organic solution; FDR, false discovery rate; XIC, extracted ion chromatogram; Hb, hemoglobin; PK, pyruvate kinase; 2D, two-dimensional; 1D, one-dimensional; LTQ, linear and have a of and are and as well as of a that a of proteins of which hemoglobin 98% of the The of and the of cytoplasmic the RBC with a to of present in are to and of the and of of and of in of and proteins and of The of the RBC that of the cell and of been well in the the and of The of red cell of the red blood cell and of a of extensive of the and proteome of red blood the cytoplasmic of the RBC, studies have on a of with to of erythrocyte and The red blood cell is erythrocyte of of and and of and and of The RBC by pyruvate and to that the cytoplasmic proteome of of a of as to present In in were by The 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