A second proteomic comparative analysis including MPs derived from the blood of a set of AA, SS, and hydroxyurea-treated (HU-treated) SS mice (n = 3). (A) Volcano plots represent average relative fold differences determined by plotting P values (–log₁₀) for each protein against the calculated fold change (log₂) difference (of that protein) in SS relative to AA microparticle peptide MS1 chromatogram intensities that were combined for each protein into protein-level data. (B) Volcano plots represent average relative fold differences determined by plotting P values (–log₁₀) for each protein against the calculated fold change (log₂) difference (of that protein) in SS relative to AA RBC lysate peptide MS1 chromatogram intensities that were combined for each protein into protein-level data. (C) Volcano plots represent average relative fold differences determined by plotting P values (–log₁₀) for each protein against the calculated fold change (log₂) difference (of that protein) in HU-treated SS relative to SS microparticle peptide MS1 chromatogram intensities that were combined for each protein into protein-level data. All peptide-spectrum matches were obtained using a strict 1% protein FDR. P values were generated by applying a t test statistic to log₂-transformed fold change values for each mouse biological condition. (D–F) Relative ion current intensity fold changes corresponding to each Hb amino acid–specific PTM from HU-treated and untreated SS mouse shear stress MPs (n = 3); measurements were determined by Hb peptide MS1 chromatogram intensities for each PTM. All peptide-spectrum matches were obtained using a strict 1% protein FDR, and P values were generated by applying a t test statistic for each mouse biological condition. (G) Representative Western blot showing comparative Hb binding in immunoprecipitated band 3 elute of RBC MPs from AA, SS, and SS treated with HU. Top panel: equal loading in band 3 protein input. Lower panel: presence of Hbα protein in the band 3 immunoprecipitate elute. (H) Average band intensities of Hb in band 3 immunoprecipitate from AA, SS, and SS + HU MPs were measured using Bio-Rad Image Lab software and expressed as relative density following normalization with corresponding band 3 protein bands. Bars represent average mean value; each dot in the bars represents an individual data point; and vertical error bars represent SEM. Student’s t test, 2-tailed, *P < 0.05 (n = 3).