The online version of this article has a Supplementary Appendix. BackgroundResults from recent, highly debated, retrospective studies raised concerns and prompted considerations about further testing the quality of long stored red blood cells from a biochemical standpoint. Design and MethodsWe performed an integrated mass spectrometry-based metabolomics and proteomics timecourse investigation on SAGM-stored red blood cells. In parallel, structural changes during storage were monitored through scanning electron microscopy. ResultsWe detected increased levels of glycolytic metabolites over the first 2 weeks of storage. From day 14 onwards, we observed a significant consumption of all metabolic species, and diversion towards the oxidative phase of the pentose phosphate pathway. These phenomena coincided with the accumulation of reactive oxygen species and markers of oxidation (protein carbonylation and malondialdehyde accumulation) up to day 28. Proteomics evidenced changes at the membrane protein level from day 14 onwards. Changes included fragmentation of membrane structural proteins (spectrin, band 3, band 4.1), membrane accumulation of hemoglobin, antioxidant enzymes (peroxiredoxin-2) and chaperones. While the integrity of red blood cells did not show major deviations at day 14, at day 21 scanning electron microscope images revealed that 50% of the erythrocytes had severely altered shape. We could correlate the scanning electron microscopy observations with the onset of vesiculation, through a proteomics snapshot of the difference in the membrane proteome at day 0 and day 35. We detected proteins involved in vesicle formation and docking to the membrane, such as SNAP alpha. ConclusionsBiochemical and structural parameters did not show significant alterations in the first 2 weeks of storage, but then declined constantly from day 14 onwards. We highlighted several parallelisms between red blood cells stored for a long time and the red blood cells of patients with hereditary spherocytosis.Key words: red blood cell, storage, mass spectrometry, proteomics, metabolomics. Haematologica 2012;97(1):107-115. doi:10.3324/haematol.2011 Citation: D'Alessandro A, D'Amici GM, Vaglio S, and Zolla L. Time-course investigation of SAGM-stored leukocyte-filtered red blood cell concentrates: from metabolism to proteomics.

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.

The changes induced in the photosynthetic apparatus of spinach (Spinacia oleracea L.) seedlings exposed to iron deficiency shortly after germination were characterized with two proteomic approaches coupled with chlorophyll and xanthophyll analysis and in vivo measurements of photosynthesis. During the first 10 d of iron deficiency the concentrations of chlorophyll b and violaxanthin were greatly reduced, but all xanthophylls recovered after 13-17 d of iron deficiency, when both chlorophylls were negatively affected. No new protein was formed in iron-deficient leaves, and no protein disappeared altogether. Photosystem I (PSI) proteins were largely reduced, but the stoichiometry of the antenna composition of PSI was not compromised. On the contrary, PSII proteins were less affected by the stress, but the specific antennae Lhcb4 and Lhcb6, Lhcb2 and its isoform Lhcb1.1 were all reduced, while the concentration of Lhcb3 increased. A strong reduction in thylakoid bending and an altered distribution pattern for the reduced PSI and PSII complexes were observed microscopically in iron-deficient leaves. Supercomplex organization was also affected by the stress. The trimeric organization of Lhcb and the dimerization of Lhca were reduced, while monomerization of Lhcb increased. However, the trimerization of Lhcb was partially recovered after 13-17 d of iron deficiency. In iron-deficient leaves, photosynthesis was strongly inhibited at different light intensities, and a high de-epoxidation status of the xanthophylls was observed, in association with a strong impairment of photochemical efficiency and an increase of heat dissipation as monitored by the non-photochemical quenching of fluorescence. All these negative effects of iron deficiency were attenuated but not fully reversed after again supplying iron to iron-deficient leaves for 7-13 d. These results indicate that iron deficiency has a strong impact on the proteomic structure of spinach photosystems and suggest that, in higher plants, adaptive mechanisms common in lower organisms, which allow rapid changes of the photosystem structure to cope with iron stress, are absent. It is speculated that the observed changes in the monomer-trimer equilibrium of major PSII antennae, which is possibly the result of xanthophyll fluctuations, is a first adaptative adjustment to iron deficiency, and may eventually play a role in light dissipation mechanisms.

The detailed analysis of these protein associations to the membrane of aged RBCs allowed Prx2 to be suggested as a potential RBC oxidative stress marker for the sake of developing new approaches in quality assurance of blood components.

The time course of the thylakoid membranes proteomic profile changes upon cadmium (Cd) addition to hydroponic Spinacia oleracea L. plants has been investigated. Two different proteomic approaches have been used: blue native gel electrophoresis followed by SDS-PAGE (2D BN-SDS-PAGE) and sucrose density gradient ultracentrifugation followed by RP-HPLC. Chlorophyll (Chl) and xanthophylls concentrations, together with ESR and real time PCR measurements, were also performed to get a complete overview of all photosystem changes. Cd only accumulated in basal leaves, that therefore were prevalently investigated for assessment of Cd induced changes. Here, Cd strongly reduced Chl concentration, especially Chl a. During the first 15 days of treatment, native electrophoresis system revealed high sensitivity of PSI to Cd, while minor effects on PSII were observed. Cytochrome b(6)/f and the ATP-synthase complex did not change following the Cd treatment. A significant reduction of antenna proteins of PSI was observed, while PSII antennae were affected to a minor extent, with exception of the isomeric Lhcb1.1 which decreased significantly already at the onset of the treatment. Some PSII core proteins were overexpressed, but showed reduced activity. No new protein was formed and no specific protein disappeared in the photosynthetic apparatus of Cd-treated leaves. Upon removal of Cd, a rapid resynthesis of total Chl and a significant resynthesis of Lhcb1.1 antenna were observed, suggesting that Cd affects specifically the photosynthetic apparatus of spinach basal leaves, replacing other metal ions inside proteins.

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