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

Florence Roux-Dalvai,Anne Gonzalez De Peredo,Carolina Simoé,Luc Guerrier,David Bouyssieé,Alberto Zanella,Attilio Citterio,Odile Burlet-Schiltz,Egisto Boschetti,Pier Giorgio Righetti,Bernard Monsarrat

doi:10.1074/mcp.m800037-mcp200

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.

Highlights

The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored
Treatment of Red Blood Cell Proteins and Preliminary Analysis—A total amount of 5730 mg of red blood cells (RBCs) cytosolic proteins was loaded on a sequence of two columns composed of the two different ligand libraries, and the captured proteins were collected by eluting each column sequentially with TUC, UCA, and hydro-organic solution (HOS) buffers
In our study of the RBC cytoplasm, we identified most of the actors of these major pathways, the enzymes involved in the most frequent metabolic RBC disorders such as pyruvate kinase, glucose-6-phosphate isomerase, or triose-phosphate isomerase

Summary

Introduction

The erythrocyte cytoplasmic proteome is composed of 98% hemoglobin; the remaining 2% is largely unexplored. We were able to detect the presence of eight different hemoglobin chains including embryonic and newly discovered globin chains 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) have a life span of approximately 120 days and are optimally adapted for oxygen and carbon dioxide as well as for proton transport They consist of a plasma membrane that envelopes a viscous concentrated (33%) solution of proteins of which hemoglobin (Hb) constitutes approximately 98% of the global proteome. These numbers are outstanding, they still fall short of expectations considering that the cytoplasmic proteomic asset would be inherited by the erythroblasts and that it is believed that living human cells should have a genetic asset of Ͼ10,000 unique gene products

Results

Discussion

Conclusion