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. Molecular & Cellular Proteomics 7: 2254 -2269, 2008.Mature red blood cells (RBCs) 1 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. The absence of nucleus and the loss of cytoplasmic organelles allow the RBC passing through narrow capillaries, with a concomitant drastic shape change, to properly accomplish its most important biological tasks. A number of other vital functions present in RBCs are related to appropriate generation and expenditure of energy. These include the following: (a) initiation and maintenance of glycolysis, (b) cation pumping against electrochemical gradients, (c) synthesis of glutathione and other metabolites, (d) nucleotide catabolism reactions, (e) maintenance of Hb iron in its functional, reduced, ferrous state, (f) protection of enzymatic and structural proteins from oxidative denaturation, and (g) preservation of membrane phospholipid asymmetry.The structure of the RBC membrane (a thin layer that constitutes less than 0.1% of the cell thickness and only 1% of its weight) has been well elucidated in the past 35 years both from the normal and pathological metabolic points of view (1, 2) and, more recently, from a structural point of view via extensive proteomics mapping (3). Regarding the cytoplasmic content of the RBC, most studies have focused on a variety of rare ...

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“…Furthermore, CPLL have been used for the analysis of human urine, human bile, platelet lysate, red blood cell lysate, chicken egg white and egg yolk, or cow whey (Castagna et al, 2005;Thulasiraman et al, 2005;Guerrier et al, 2006Guerrier et al, , 2007aGuerrier et al, , 2007bD'Ambrosio et al, 2008;Roux-Dalvai et al, 2008;Boschetti and Righetti, 2009;D'Amato et al, 2009;Farinazzo et al, 2009). In all these studies, a much larger number of proteins could be identified when using CPLL, with many of the proteins being described for the first time in the respective sample.…”

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“…In this context, it appeared that the sequential use of a carboxylated library captured additional species that escaped from the first, aminoterminus, library. This result was obtained from platelet extracts (Guerrier et al, 2007a), from human bile (Guerrier et al, 2007b), as well as from red blood cell lysates (Roux-Dalvai et al, 2008). As an average, the additional proteins caught by the second library represented between 20% and 30% of the total number.…”

“…This goal appears as reachable-at least for simple cells-not only from prokaryotes but also from eucaryotes, such as for instance red blood cells. In a recent presentation, Roux-Dalvai et al (2008) showed that the total count of gene products, that represents the 2% minority RBC cytoplasmic proteome, reached 1,578 unique proteins; U: that is a truly outstanding count, considering that a RBC is a much simplified cell (no nucleus, no other organelles) than any other human cell lines, believed to comprise a genetic asset of >12,000 unique gene products (a mere estimate, because at present, no study has been able to explore to full proteome of any human cell). What is the meaning of such a large body of proteins in the RBC cytoplasm?…”

“…In another series of trials, Roux-Dalvai et al (2008) described the addition of 100, 300 or 1000 pmol of yeast alcohol dehydrogenase (ADH) to 680 mg of red blood cell lysate, prior to processing with a 0.5 mL of a CPLL column. After extensive washing, the captured proteins were eluted with a 9 M urea solution, acidified with citric acid to pH 3.3.…”

“…Its application to proteomics, though, is relatively recent, because the first report appeared only 3 years ago (Thulasiraman et al, 2005). A flurry of applications soon followed; for example, in urine (Castagna et al, 2005), serum Sennels et al, 2007), human platelets (Guerrier et al, 2007a), red blood cell (Roux-Dalvai et al, 2008), bile (Guerrier et al, 2007b), and recombinant DNA product Antonioli et al, 2007) analyses. This technology is presently commercially available under the trade name of ProteoMiner.…”