Hemospan is an acellular hemoglobin-based oxygen therapeutic in clinical trials in Europe and the United States. The product is prepared by site-specific conjugation of maleimide-activated poly(ethylene) glycol (PEG, MW approximately 5500) to human oxyhemoglobin through maleimidation reactions either (1) directly to reactive Cys thiols or (2) at surface Lys groups following thiolation using 2-iminothiolane. The thiolation/maleimidation reactions lead to the addition of approximately 8 PEGs per hemoglobin tetramer. Identification of PEG modified globins by SDS-PAGE and MALDI-TOF reveals a small percentage of protein migrating at the position for unmodified globin chains and the remaining as separate bands representing globin chains conjugated with 1 to 4 PEGs per chain. Identification of PEG modification sites on individual alpha and beta globins was made using reverse-phase HPLC, showing a series of alpha globins conjugated with 0 to 3 PEGs and a series of beta globins conjugated with 0 to 4 PEGs per globin. Mass analysis of tryptic peptides from hemoglobin thiolated and maleimidated with N-ethyl maleimide showed the same potential sites of modification regardless of thiolation reaction ratio, with seven sites identified on beta globins at beta8, beta17, beta59, beta66, beta93, beta95, and beta132 and three sites identified on alpha globins at alpha7, alpha16, and alpha40.