Synthesis and Characterization of Protein CEP Adducts

All CEP adducts were synthesized as described [10] (link). The dipeptide, Ac-Gly-Lys-OMe, was obtained from BACHEM; HSA from AlbuminBio; MSA from AlbuminBio or Sigma; phosphatidyl ethanolamine was from Sigma. Controls for protein CEP adducts included untreated protein (CTL1) and treated unadducted protein (CTL2). The latter control was processed in the same synthesis procedure as that for CEP adducts, except 4,7-dioxoheptanoic acid 9-fluorenylmethyl ester was left out to avoid the covalent addition of CEP moiety. Protein CEP adducts, after final dialysis in PBS, were quantified by the Bradford assay and tested for endotoxins with the Endosafe-PTS kit (Charles River). For storage, samples were filtered through 0.2 µm, divided aseptically in 1-mL aliquots, and stored at −80 C.