Oxidation of functional GAPDH residues and GAPDH vesiculation into supernatants. (A) A structural representation of GAPDH active site pocket, with Cys152 and His179 modified as to show 2 confidently assigned redox modifications (Cys to DHA; His to 2-oxo-His). (B) Ratios of NEM-tagged or IAM-tagged Cys152 (green and red continuous lines, respectively) vs total spectral matches for GAPDH in total RBC extracts at storage days 2, 21, and 42. Results represent a readout of unmodified or reversibly modified Cys152 GAPDH at lysis, respectively. (C) Absolute quantitation of GAPDH in stored RBC supernatants through QconCAT analysis. (D) Semiquantitative determination of oxidative modifications to Cys152 of GADPH are shown, calculated by determining the ratios of modified/total occurrences of confidently observed (P < .05) GAPDH peptide IISNASCTTNCLAPLAK, divided by the total number of all peptides confidently assigned for GAPDH.