Inherited hemolytic anemia (spherocytosis or elliptocytosis) is one of the most common inherited diseases. While mild to severe inherited hemolytic anemias can arise from defects in the red blood cell (RBC) membrane skeleton, fundamental questions remain unanswered surrounding the clinical variability of known red blood cell mutations. To identify candidate proteins involved in hemolytic anemia pathophysiology, we utilized a label-free comparative approach to detect differences in RBCs from normal and beta-adducin (ADD2KO) knock-out mice. For each genotype, whole blood was taken from independent biological replicates and RBCs were purified using cellulose acetate chromatography. The isolated RBCs were lysed to generate RBC ghosts whose protein complements were digested with trypsin. For each biological replicate, three replicate runs utilizing 0.5 μg digested protein were performed via microcapillary liquid chromatography coupled with tandem mass spectrometry. Using the recently developed software package CRAWDAD, we detected 7 proteins that were decreased and 31 proteins with a greater abundance in the beta-adducin knock-out RBC ghosts. Differences were detected for previously known membrane skeleton components, including the predicted absence of beta-adducin and decrease in alpha-adducin. The actin binding protein capping protein (actin filament) muscle Z-line alpha 1 was increased along with ankyrin 1, spectrin alpha, tropomyosin, and glycophorin C. Interestingly, differences were also detected for the protein oxidative stress-responsive 1 and the uncharacterized protein C10orf58 which contains a thioredoxin motif. While the protein differences span a broad range of cellular processes, some of the proteins are attractive candidates for modifiers of disease severity. This label free approach is the first demonstration of comprehensively analyzing wild type versus kockout mice that circumvents the need for metabolic or chemical labeling with isotopes. This new approach adds a valuable tool to the list of standard assays for analysis of RBCs in cases of hereditary hemolytic anemia.

Disclosures: No relevant conflicts of interest to declare.

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