Proteomic analysis of RBC membrane protein degradation during blood storage.
Two-dimensional gel electrophoresis and mass spectrometry were used to identify protein profile changes in red blood cell membranes stored over time under atmospheric oxygen, in the presence or absence of protease inhibitors. New spots with lower molecular masses, ranging between 7 and 15 kDa were observed during the first 7 days storage, while over time, further fragments and high-molecular-mass aggregates appeared, seen as a smearing in the upper part of the gel. Some of the protein changes turned out to be shifts in isoelectric point, as a consequence of chemical oxidations. All these new spots were generated as a result of protein attack by reactive oxygen species (ROS). Protein identification revealed that most of the modified proteins are located in the cytoskeleton. During the first 7 days of storage, oxidative degradation was observed prevalently in band 4.2, to a minor extent in bands 4.1 and 3, and in spectrin. After 14 days, there were new fragments from beta-actin, glyceraldehyde-3-phosphate dehydrogenase, band 4.9, and ankyrin, among others. Preliminary protein-protein cross-linked products, involving alpha and beta spectrin, were also detected. The cross-linked products increased over time. Protein degradation was greatly reduced when oxygen was removed and blood was stored under helium. Interestingly, very few spots were related to enzyme activity, and they were more numerous when oxygen was present, suggesting that some proteases may be oxygen-dependent.
D'Amici
Gian Maria
GM
Department of Environmental Sciences, Tuscia University, Viterbo, Italy.
Rinalducci
Sara
S
Zolla
Lello
L
D000595:Amino Acid Sequence; D001798:Blood Proteins / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D006801:Humans; D008969:Molecular Sequence Data; D018384:Oxidative Stress / Q000502:physiology; D010455:Peptides / Q000032:analysis*; D020543:Proteome / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods
D001798:Blood Proteins; D010455:Peptides; D020543:Proteome; D017382:Reactive Oxygen Species