Tandem MS/MS analysis of band 3 protein from young and old erythrocytes
AdvisorMarchalonis, John J.
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PublisherThe University of Arizona.
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AbstractEvery day, billions of senescent human erythrocytes are removed from the circulation. The mechanism of recognition involves the formation of a neo-antigen on senescent cells, which binds autologous immunoglobulin and targets the senescent cell for phagocytosis. This neo-antigen is derived from an existing integral membrane protein, band 3. The molecular mechanisms underlying the formation of this neo-antigen during the aging process are poorly understood, but oxidative damage is suggested to be a critical event. Several post-translational modifications have been associated with aging that may contribute to altered antigenicity of the band 3 molecule, either directly by forming a covalent modification that contributes to the neo-antigen epitope or indirectly by altering the conformation of the protein, exposing hidden epitopes. Tandem mass spectral analysis was performed on tryptic digests of the band 3 protein from young and old erythrocytes. Six oxidations of methionyl residues were detected, one of which lies adjacent to a region of band 3 proposed to form an epitope of the neo-antigen and one of which lies between proposed antigenic regions. Studies of vitamin E deficiency and supplementation strongly support oxidation as a pivotal event in alteration of band 3 with aging, and the oxidized methionines identified in this study may represent the critical sites of damage. A possible deamidation was also identified in an antigenic region of band 3. Deamidation is suggested to serve as a molecular timeclock for proteins, and conversion of a glutamine to a glutamic acid may alter the antigenicity at this critical region of band 3. This work represents the first application of tandem MS/MS methodologies to a large integral membrane protein. Forty-four of the 75 band 3 tryptic peptides were characterized, covering 61% of the band 3 polypeptide. Many of the tryptic peptides did not meet criteria for proper peptide analysis. Of the remaining peptides, 95.3% of the band 3 molecule was characterized, with 55% sequenced by MS/MS. Since only partial sequence information is expected for this type of analysis, these percentages represent a tremendously successful application of the technique.
Degree ProgramGraduate College
Microbiology and Immunology