title: Hemoglobin oxidation at functional amino acid residues during routine storage of red blood cells.
abstract: BACKGROUND
  Routine storage of red blood cells (RBCs) results in the progressive accumulation of storage lesions. While the clinical relevance of these lesions is still a matter of debate, alterations to RBC morphology and biochemistry, especially in terms of energy and redox homeostasis, are likely to affect RBC physiology and functionality at a minimum. Identification of oxidative modifications that accumulate on key RBC proteins will help bridge the gap between storage induced alterations and post-transfusion RBC viability.
  
  
  METHODS
  Five AS-3 units were analyzed during routine storage via one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis-nano-high-performance liquid chromatography coupled online with tandem mass spectrometry and advanced database searches.
  
  
  RESULTS
  We identified oxidative modifications to functional residues of hemoglobin (Hb) beta chain, including proximal histidine, cysteine beta 94 (counting initiator methionine in the sequence), and histidine 144. Semiquantitative analysis indicates that up to approximately 20% of total Hb could be targeted by these oxidative modifications that are overlooked by standard proteomics approaches using routine database search conditions. Progressive accumulation of oxidized residues in stored RBCs and selective accumulation in vesicles was observed, further substantiating the hypothesis that vesiculation represents a self-protective mechanism in ageing RBCs.
  
  
  CONCLUSIONS
  Several of the oxidized residues identified play well-established roles in heme iron coordination, 2,3-diphosphoglycerate binding, and nitric oxide homeostasis. Further functional and structural studies are necessary to determine possible associations between these modifications and impaired gas transport homeostasis in RBCs from old units.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.
authors.forename: Matthew
authors.initials: M
authors.lastname: Wither
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.
authors.forename: Monika
authors.initials: M
authors.lastname: Dzieciatkowska
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.
authors.forename: Travis
authors.initials: T
authors.lastname: Nemkov
authors.affiliation: Current address: Rinat-Pfizer Inc., South San Francisco, California.
authors.forename: Pavel
authors.initials: P
authors.lastname: Strop
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.
authors.forename: Angelo
authors.initials: A
authors.lastname: D'Alessandro
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Colorado Denver-Anschutz Medical Campus, Aurora, Colorado.
authors.forename: Kirk C
authors.initials: KC
authors.lastname: Hansen
mesh_terms: D001793:Blood Preservation; D004912:Erythrocytes / Q000737:chemistry* / Q000166:cytology; D005260:Female; D006454:Hemoglobins / Q000737:chemistry*; D006801:Humans; D008297:Male; D010084:Oxidation-Reduction
chemical_list: D006454:Hemoglobins