Assessment of the Red Cell Proteome of Young Patients with Unexplained Hemolytic Anemia by Two-Dimensional Differential In-Gel Electrophoresis (DIGE)

Abstract

Erythrocyte cytosolic protein expression profiles of children with unexplained hemolytic anemia were compared with profiles of close relatives and controls by two-dimensional differential in-gel electrophoresis (2D-DIGE). The severity of anemia in the patients varied from compensated (i.e., no medical intervention required) to chronic transfusion dependence. Common characteristics of all patients included chronic elevation of reticulocyte count and a negative workup for anemia focusing on hemoglobinopathies, morphologic abnormalities that would suggest a membrane defect, immune-mediated red cell destruction, and evaluation of the most common red cell enzyme defects, glucose-6-phosphate dehydrogenase and pyruvate kinase deficiency. Based upon this initial workup and presentation during infancy or early childhood, four patients classified as hereditary nonspherocytic hemolytic anemia (HNSHA) of unknown etiology were selected for proteomic analysis. DIGE analysis of red cell cytosolic proteins clearly discriminated each anemic patient from both familial and unrelated controls, revealing both patient-specific and shared patterns of differential protein expression. Changes in expression pattern shared among the four patients were identified in several protein classes including chaperons, cytoskeletal and proteasome proteins. Elevated expression in patient samples of some proteins correlated with high reticulocyte count, likely identifying a subset of proteins that are normally lost during erythroid maturation, including proteins involved in mitochondrial metabolism and protein synthesis. Proteins identified with patient-specific decreased expression included components of the glutathione synthetic pathway, antioxidant pathways, and proteins involved in signal transduction and nucleotide metabolism. Among the more than 200 proteins identified in this study are 21 proteins not previously described as part of the erythrocyte proteome. These results demonstrate the feasibility of applying a global proteomic approach to aid characterization of red cells from patients with hereditary anemia of unknown cause, including the identification of differentially expressed proteins as potential candidates with a role in disease pathogenesis.

Plasmodium vivax trophozoite-stage proteomes

“…The proteome reported here is comparable in size to the current human RBC proteome of 2289 unique proteins [75]. Proteomes of the mature RBC, which is anuclear, contain only a few ribosomal proteins [76,77], which are thought to be left over from the reticulocyte development stage [76]. Our identification of numerous ribosomal proteins is thus consistent with the P. vivax infected host RBCs being reticulocytes and not mature RBCs.…”

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 …”

“…A limitation of the study was the number of samples analyzed; in similar studies of human patients with hereditary spherocytosis, small numbers of control subjects are also described with an emphasis on the quantitative alterations in proteins in these patients . In addition, lack of replication of the results prohibits statistical evaluation of the results and the fold changes reported should be interpreted in this light.…”

“…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 …”

“…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 . For protein amounts that increased in the affected dog compared with that of the control dog, DAVID analysis revealed an enrichment of proteins associated with the functional annotation terms “citrate cycle (TCA cycle)” and “oxidative phosphorylation,” while proteins that decreased were associated with the functional annotation terms “proteasome,” “chaperonin‐containing T‐complex,” and “intermediate filament protein” (Table ).…”