Sickle-cell disease investigated by computational proteomics approaches

Sickle-cell disease (SCD) is a clinically heterogeneous autosomal recessive monogenic disorder characterized by recurrent episodes of severe haemolysis, vaso-occlusion and infection. Proteomics promises to offer novel unbiased molecular insights into the pathophysiology of SCD. The objective of this project is to analyze by bioinformatic tools mass spectrometry (MS) proteomics raw data, which has been generated by INSA’s Proteomic Laboratory in order to investigate proteome changes that might be related with SCD vaso-occlusion exacerbation. The MS raw data included in this study was generated by shotgun proteomic analysis on red blood cell (RBC) samples from six child SCD patients at steady-state and vaso-occlusion exacerbation episode and five child control subjects. The RBC samples, the haemoglobin depleted-cytoplasmic fraction and membrane fraction, were analysed by proteomic discovery-based approach using the ESI-LQT Orbitrap XL (Thermo) mass spectrometer. The generated MS raw data files were analysed by the PatternLab for Proteomic 4.0 bioinformatic platform for protein identification and extracted-ion chromatograms (XICs)-based label-free relative quantification. The following “golden parameters” were applied to obtain reliable and trustworthy data: one unique peptide at least should be considered to infer a protein identification, and the identified proteins should be present at least in 80% of the studied groups/conditions. 250 proteins were identified, and the respective normalized ion abundance factor was compared by using the Wilcoxon-T non-parametric statistical test. The differentially expressed proteins in crisis as compared to steady-state (p-value ≤ 5%) were investigated in the light of the Gene Ontology (GO) knowledge base (Database for Annotation, Visualization and Integrated Discovery – DAVID) and Reactome database for protein integration into signaling pathways with biological meaning. The most relevant results indicated that 3 cytoplasmic proteins (Dematin, Moesin and Protein S100-A4) and 9 membrane proteins (Eosinophil cationic protein, Bone marrow proteoglycan, Voltage-dependent anion-selective channel protein 1, Voltage-dependent anion-selective channel protein 2, Reticulon-3, Carbonic anhydrase 2, Haemoglobin subunit alpha, Haemoglobin subunit delta and Eosinophil peroxidase) showed significantly differential expression in crisis as compared to steady-state. Haemoglobin subunit alpha and Carbonic anhydrase 2 (CA2) have been reported as involved in important pathways related with O2/CO2 exchange in erythrocytes. Sickle cell crisis is frequently related to infection, involving Reticulon-3 and Haemoglobin subunit alpha. These proteins were identified to be modulated in the RBC membrane fraction from SCD patients at crisis. The most relevant proteins identified by these computational approaches will be selected for further biochemical verification by using SWATH-MS and/or Western blot methods. These proteins may be promising candidate early biomarkers to identify SCD patients at risk for vaso-occlusion crisis.Fundação para a Ciência e a Tecnologia partially financing this project (Grant PIC/IC/83084/2007 and Doctoral Fellowship - SFRH/BPD/31209/2006).N/