Mass spectrometric characterization of human hemoglobin adducts formed in vitro by hexahydrophthalic anhydride

Primary structural information of anhydride binding to endogenous proteins is of interest in order to determine the mechanism causing the type-I allergy seen in many anhydride-exposed workers. In addition, studies on specific protein adducts may generate new methods for biological monitoring. In this study, the binding of hexahydrophthalic anhydride (HHPA) to human hemoglobin (Hb) in vitro was investigated. The in vitro synthesized conjugates were analyzed using a hybrid quadrupole-time-of-flight mass spectrometer (Q-TOF) with electrospray ionization (ESI) to determine the number of HHPA adducts per Hb molecule. Structural information on the locations of the adducts was obtained through nanospray Q-TOF, liquid chromatography-ESI mass spectrometric analysis, and gas chromatography/mass spectrometric analysis of Pronase E and tryptic digests. Up to six adducts were found on the alpha-chain and five on the beta-chain. The HHPA-adducts were localized to the N-terminal valine of the alpha- and beta-chains of Hb and to lysine residues at positions 7, 11, 16, and 40 of the alpha-chain and 8, 17, 59, 66, and 144 of the beta-chain. These results will constitute a basis for studies on structure-activity relationships as well as for development of methods for biological monitoring of acid anhydrides.