Comprehensive identification of erythrocyte membrane protein deficiency by 2D gel electrophoresis based proteomic analysis in hereditary elliptocytosis and spherocytosis

Abstract

Purpose Sodium dodecyl sulphate‐polyacrylamide gel electrophoresis (1DE) may reveal qualitative or quantitative defects in red blood cell (RBC) membrane proteins, two‐dimensional gel electrophoresis (2DE) can be used for determination of the protein changes caused by the disease process in a relatively high‐throughput manner, because it permits an analysis of thousands of modified or unmodified proteins simultaneously. The principal aim of this study was to compare hereditary elliptocytosis (HE), hereditary spherocytosis (HS), and control RBC membrane protein profiles and identify proteins as a clinical marker by the sensitive methods. Experimental design RBC membrane proteins of HE, HS, and control groups were compared by 2DE and matrix‐assisted laser desorption/ionization‐time of flight (MALDI‐TOF) MS analysis was obtained using peptide mass fingerprint for protein identification. Results Twenty proteins were identified with peptide mass fingerprint analysis and different expression levels were found in HE, HS, and controls for some proteins that includes three biomarker proteins (ankyrin, spectrin, band 3) that may have prognostic or predictive importance. Conclusions and clinical relevance 2DE of RBC proteins is a potentially valuable method for studying heritable disorders such as HE and HS. By identifying a deficiency in membrane proteins associated with the RBC cytoskeleton, the diagnosis of HE and HS can be confirmed.

Assessment of the red blood cell proteome in a dog with unexplained hemolytic anemia

“…Interestingly, this same protein derangement pattern was previously reported in people with primary band 3 deficiencies . The proteomic approach used here is recognized as a diagnostic approach when investigating hereditary spherocytosis in people; however, to the authors’ knowledge, this technique has not been previously applied as a diagnostic approach in dogs. Proteomics also uncovered protein level differences between the affected and control dogs, highly consistent with previous descriptions of reticulocytoses and ongoing hemolyses …”

“…Recently published guidelines recommend the use of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to explore alterations to the erythrocyte protein profiles, followed by sequence analysis of the membrane protein genes when the protein results do not explain the clinical outcome or mode of inheritance . In tandem with these recommendations, high‐throughput proteomic analysis has been applied to patients presenting with hereditary hemolysis and in some cases identified hypothesized protein deficiencies …”

“…A total of 408 proteins were identified with high confidence (defined by at least 2 peptide matches), of which 252 proteins were found to be increased or decreased more than 2‐fold in the patient compared with that of the control (Table ). In this study, a 2‐fold change was taken as the cutoff value to identify proteins that had significantly altered amounts . This approach allowed exploration of the protein differences detected in the presence of a reticulocytosis and hemolysis as previously described .…”

“…6 In tandem with these recommendations, high-throughput proteomic analysis has been applied to patients presenting with hereditary hemolysis and in some cases identified hypothesized protein deficiencies. 5,7 Red blood cells, aside from reticulocytes, are particularly attractive for proteomic analysis due to their lack of internal membranebound organelles and nucleus, resulting in limited complexity.…”

Erythrocyte and platelet proteomics in hematological disorders

“…Rabilloud established more sensitive methods of gel staining leading to visualizing greater number of protein spots in 2DE [10]. Following this, few gel-based studies on erythrocyte membrane came up, describing protein deregulation in hereditary spherocytosis [11][12][13][14][15], sickle cell anemia [16][17][18], and hemoglobin E ␤ (HbE␤) thalassemia [19]. Few novel proteins were identified in malaria parasite infected erythrocyte membrane [20].…”