Label-free quantitation of phosphopeptide changes in erythrocyte membranes: towards molecular mechanisms underlying deformability alterations in stored red blood cells | Haematologica

摘要

While the probability of troublesome adverse effects related to the transfusion of older red blood cell (RBC) units is still a matter of debate and of clinical investigation, what is now known for certain is that blood storage affects the biochemical and biological properties of RBCs. The accumulating changes, collectively known as “storage lesions”, include alterations to either functionality (essentially metabolism and oxygen delivery capacity), or morphology (transition from a discoid to a spherocytic phenotype). These latter are mostly irreversible and result in a more rigid cell structure, with cytoskeleton disorders and perturbation of membrane protein interactions; therefore, they are likely the most responsible for reducing transfusion efficacy. Protein phosphorylation is known to be one of the most important and better-studied posttranslational modifications that affect protein-protein binding interfaces. Interestingly, all components of the red cell membrane skeleton (except actin) are phosphoproteins.1 Past research has demonstrated that their phosphorylation is involved in the mechanical properties of the erythrocyte membrane.2–4 However, there have been no studies at all on the phosphorylation events occurring during in vitro RBC aging. Based on these considerations, we aimed to investigate the phosphorylation status of erythrocyte membranes while undergoing blood storage for transfusion purposes by means of phosphoproteomics technologies. To this end, we decided to apply a gel-free shotgun proteomics approach to obtain qualitative phosphorylation site mapping of RBC ghosts. Specifically, packed RBCs were lysed with 9 vol of cold 5 mM phosphate buffer pH 8.0 containing 1 mM EDTA, 1mM phenylmethanesulfonyl fluoride (PMSF) and phosphatase inhibitor cocktails (P5726, P0044 Sigma-Aldrich). Two hundred micrograms of red cell membrane proteins were subjected to in-solution tryptic digestion5 followed by selective pre-enrichment of phosphorylated peptides through TiO2 affinity chromatography microcolumns.6 Eluted phosphopeptides were then analyzed by LC-MS/MS with both electron transfer dissociation (ETD) and neutral-loss triggered MS3 in collision-induced dissociation (CID), as previously reported.7 Experiments were performed in triplicate at 0-day storage time with five leuko-reduced CPD-SAGM RBC units (biological replicates) collected from different donors. Results are shown in Table 1. As expected, pTyr occurrence was very low, but in agreement with the estimated percentage,8 while numerous Ser/Thr phosphopeptides were identified. Five of these were chosen to be quantitatively monitored during storage (at 0, 21 and 35 days); selection criteria are described in Table 1. The analytical strategy adopted for the quantitation consisted in a targeted approach because we only quantified (by LC-MS) those individual peptide ions of interest that had been detected and identified previously in data-dependent LC-MS/MS experiments. In detail, targeted quantification was performed by adopting a conventional label-free MS-based workflow relying on the calculation of the extracted ion chromatogram (EIC) peak height from LC-MS runs.9,10 To minimize technical variability, each sample (i.e. 200 μg of digested erythrocyte membrane proteins) were spiked with 25 fmol/μg of bovine α-casein digest prior to TiO2 enrichment. Among the α-casein phosphopeptides detected, the one showing the lower coefficient of variation (CV) was chosen as internal standard for data normalization (i.e. YKVPQLEIVPNSpAEER, m/z 976.40, CV = 14%). Figure 1 shows graphs obtained by plotting the normalized ion intensities of each phosphopeptide versus storage time. Measurements of phosphopeptide intensity variation across all replicates for each condition resulted in CV values ranging from 16% to 40%. Quantitative data have been validated by multiple reaction monitoring (MRM)-based experiments (data not shown). Although each phosphopeptide showed a distinctive tre