Red blood cell deformation induces measurable changes in the phosphoproteome. (A) Experimental design for proteomic comparison: RBCs and cultured reticulocytes were subjected to flow in a microsphiltration system and lysed before, during, and after passage. The lysates were equalized through hemoglobin quantification using Drabkin’s reagent and analyzed through NanoLC-MS/MS. Cells were fixed and imaged under brightfield at all 3 stages, showing the extent of their deformation within the system and their recovery after passage. Scale bars, 10 μm. (Microsphiltration schematic adapted from Duez et al.⁴⁸ ) (B) Venn diagram of differentially phosphorylated proteins with ≥50% detection across all samples in RBCs (left, n = 63 with 33 modifications detected exclusively in cells mid- and postdeformation) and reticulocytes (right, n = 226 with 55 modifications detected exclusively in cells mid- and postdeformation). (C) Top-scoring protein-protein interaction network from the joint dataset of mid- and postdeformation exclusive phosphoproteins in reticulocytes, as predicted by STRING.⁴⁹  Two main subsets of proteins were present in the network, kinases (marked in red) and vesicle transport-related proteins (marked in dark blue). (D) List of phosphoproteins exclusive to the RBC mid-deformation and mid-/postintersection datasets. Count indicates the number of observed instances of the phosphopeptide (n = 3); the phosphopeptides were sorted by the sum of their counts in both datasets and filtered for their presence in at least 2 samples per condition. PhosphoSite location indicates the predicted location of the phosphorylation site in the protein sequence (as predicted by SEQUEST and/or PhosphoSitePlus data⁵⁰ ), as well as the effect of its phosphorylation if described in low-throughput studies according to PhosphoSitePlus.⁵⁰  (E) List of phosphoproteins exclusive to the reticulocyte mid-deformation and mid-/postintersection datasets (n = 4). Dataset analysis, filtering, and annotation were performed as previously described. (F) List of phosphoproteins present among both reticulocyte and RBC mid-/postdeformation datasets, with no significant detection in the predeformation dataset; dataset analysis, filtering, and annotation were performed as previously described.