Methylation of protein aspartates and deamidated asparagines as a function of blood bank storage and oxidative stress in human red blood cells

Transfusion. 2018 Dec;58(12):2978-2991. doi: 10.1111/trf.14936. Epub 2018 Oct 12.


Background: Being devoid of de novo protein synthesis capacity, red blood cells (RBCs) have evolved to recycle oxidatively damaged proteins via mechanisms that involve methylation of dehydrated and deamidated aspartate and asparagine residues. Here we hypothesize that such mechanisms are relevant to routine storage in the blood bank.

Study design and methods: Within the framework of the REDS-III RBC-Omics (Recipient Epidemiology Donor Evaluation Study III Red Blood Cell-Omics) study, packed RBC units (n = 599) were stored under blood bank conditions for 10, 23, and 42 days and profiled for oxidative hemolysis and time-dependent metabolic dysregulation of the trans-sulfuration pathway.

Results: In these units, methionine consumption positively correlated with storage age and oxidative hemolysis. Mechanistic studies show that this phenomenon is favored by oxidative stress or hyperoxic storage (sulfur dioxide >95%), and prevented by hypoxia or methyltransferase inhibition. Through a combination of proteomics approaches and 13 C-methionine tracing, we observed oxidation-induced increases in both Asn deamidation to Asp and formation of methyl-Asp on key structural proteins and enzymes, including Band 3, hemoglobin, ankyrin, 4.1, spectrin beta, aldolase, glyceraldehyde 3-phosphate dehydrogenase, biphosphoglycerate mutase, lactate dehydrogenase and catalase. Methylated regions tended to map proximal to the active site (e.g., N316 of glyceraldehyde 3-phosphate dehydrogenase) and/or residues interacting with the N-terminal cytosolic domain of Band 3.

Conclusion: While methylation of basic amino acid residues serves as an epigenetic modification in nucleated cells, protein methylation at carboxylate side chains and deamidated asparagines is a nonepigenetic posttranslational sensor of oxidative stress and refrigerated storage in anucleated human RBCs.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Asparagine / metabolism*
  • Aspartic Acid / metabolism*
  • Blood Banks*
  • Blood Preservation*
  • Erythrocytes / cytology
  • Erythrocytes / metabolism*
  • Humans
  • Methylation
  • Oxidative Stress*
  • Protein Processing, Post-Translational*
  • Proteomics
  • Time Factors


  • Aspartic Acid
  • Asparagine