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.
| Original language | English |
|---|---|
| Pages (from-to) | 2254-2269 |
| Number of pages | 16 |
| Journal | Molecular and Cellular Proteomics |
| Volume | 7 |
| Issue number | 11 |
| DOIs | |
| Publication status | Published - Nov 2008 |
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ASJC Scopus subject areas
- Biochemistry
- Molecular Biology
- Analytical Chemistry
Cite this
Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry. / Roux-Dalvai, Florence; de Peredo, Anne Gonzalez; Simó, Carolina; Guerrier, Luc; Bouyssié, David; Zanella, Alberto; Citterio, Attilio; Burlet-Schiltz, Odile; Boschetti, Egisto; Righetti, Pier Giorgio; Monsarrat, Bernard.
In: Molecular and Cellular Proteomics, Vol. 7, No. 11, 11.2008, p. 2254-2269.Research output: Contribution to journal › Article
}
TY - JOUR
T1 - Extensive analysis of the cytoplasmic proteome of human erythrocytes using the peptide ligand library technology and advanced mass spectrometry
AU - Roux-Dalvai, Florence
AU - de Peredo, Anne Gonzalez
AU - Simó, Carolina
AU - Guerrier, Luc
AU - Bouyssié, David
AU - Zanella, Alberto
AU - Citterio, Attilio
AU - Burlet-Schiltz, Odile
AU - Boschetti, Egisto
AU - Righetti, Pier Giorgio
AU - Monsarrat, Bernard
PY - 2008/11
Y1 - 2008/11
N2 - 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.
AB - 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.
UR - http://www.scopus.com/inward/record.url?scp=43149086780&partnerID=8YFLogxK
UR - http://www.scopus.com/inward/citedby.url?scp=43149086780&partnerID=8YFLogxK
U2 - 10.1074/mcp.M800037-MCP200
DO - 10.1074/mcp.M800037-MCP200
M3 - Article
C2 - 18614565
AN - SCOPUS:43149086780
VL - 7
SP - 2254
EP - 2269
JO - Molecular and Cellular Proteomics
JF - Molecular and Cellular Proteomics
SN - 1535-9476
IS - 11
ER -