Tandem MS/MS analysis of band 3 protein from young and old erythrocytes

Persistent Link:
http://hdl.handle.net/10150/282574
Title:
Tandem MS/MS analysis of band 3 protein from young and old erythrocytes
Author:
Adams, Charlotte Lynne, 1971-
Issue Date:
1997
Publisher:
The University of Arizona.
Rights:
Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.
Abstract:
Every 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.
Type:
text; Dissertation-Reproduction (electronic)
Keywords:
Biology, Cell.; Chemistry, Analytical.
Degree Name:
Ph.D.
Degree Level:
doctoral
Degree Program:
Graduate College; Microbiology and Immunology
Degree Grantor:
University of Arizona
Advisor:
Marchalonis, John J.

Full metadata record

DC FieldValue Language
dc.language.isoen_USen_US
dc.titleTandem MS/MS analysis of band 3 protein from young and old erythrocytesen_US
dc.creatorAdams, Charlotte Lynne, 1971-en_US
dc.contributor.authorAdams, Charlotte Lynne, 1971-en_US
dc.date.issued1997en_US
dc.publisherThe University of Arizona.en_US
dc.rightsCopyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author.en_US
dc.description.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.en_US
dc.typetexten_US
dc.typeDissertation-Reproduction (electronic)en_US
dc.subjectBiology, Cell.en_US
dc.subjectChemistry, Analytical.en_US
thesis.degree.namePh.D.en_US
thesis.degree.leveldoctoralen_US
thesis.degree.disciplineGraduate Collegeen_US
thesis.degree.disciplineMicrobiology and Immunologyen_US
thesis.degree.grantorUniversity of Arizonaen_US
dc.contributor.advisorMarchalonis, John J.en_US
dc.identifier.proquest9817351en_US
dc.identifier.bibrecord.b38269351en_US
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