title: Targeted quantitative phosphoproteomic analysis of erythrocyte membranes during blood bank storage.
abstract: One of the hallmarks of blood bank stored red blood cells (RBCs) is the irreversible transition from a discoid to a spherocyte-like morphology with membrane perturbation and cytoskeleton disorders. Therefore, identification of the storage-associated modifications in the protein-protein interactions between the cytoskeleton and the lipid bilayer may contribute to enlighten the molecular mechanisms involved in the alterations of mechanical properties of stored RBCs. Here we report the results obtained analyzing RBCs after 0, 21 and 35 days of storage under standard blood banking conditions by label free mass spectrometry (MS)-based experiments. We could quantitatively measure changes in the phosphorylation level of crucial phosphopeptides belonging to β-spectrin, ankyrin-1, α-adducin, dematin, glycophorin A and glycophorin C proteins. Data have been validated by both western blotting and pseudo-Multiple Reaction Monitoring (MRM). Although each phosphopeptide showed a distinctive trend, a sharp increase in the phosphorylation level during the storage duration was observed. Phosphopeptide mapping and structural modeling analysis indicated that the phosphorylated residues localize in protein functional domains fundamental for the maintenance of membrane structural integrity. Along with previous morphological evidence acquired by electron microscopy, our results seem to indicate that 21-day storage may represent a key point for the molecular processes leading to the erythrocyte deformability reduction observed during blood storage. These findings could therefore be helpful in understanding and preventing the morphology-linked mechanisms responsible for the post-transfusion survival of preserved RBCs.
authors: Department of Ecological and Biological Sciences, University of Tuscia, Viterbo, Italy.
authors: Sara
authors: S
authors: Rinalducci
authors: Valentina
authors: V
authors: Longo
authors: Luigi R
authors: LR
authors: Ceci
authors: Lello
authors: L
authors: Zolla
mesh_terms: D001793:Blood Preservation; D001798:Blood Proteins / Q000032:analysis* / Q000737:chemistry; D015153:Blotting, Western; D004910:Erythrocyte Membrane / Q000737:chemistry*; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000097:blood* / Q000737:chemistry; D008958:Models, Molecular; D010750:Phosphoproteins / Q000097:blood* / Q000737:chemistry; D020543:Proteome / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods
chemical_list: D001798:Blood Proteins; D008565:Membrane Proteins; D010750:Phosphoproteins; D020543:Proteome
keywords: MS-based quantitation; blood storage; membrane skeletal proteins; phosphorylation; red cells