Identification of modification sites on human serum albumin and human hemoglobin adducts with houttuynin using liquid chromatography coupled with mass spectrometry.

Houttuynin, a β-keto aldehyde compound, is the major active ingredient in herba houttuyniae injection. The injection was once used as an anti-inflammatory drug associated with occasional serious hypersensitivity reactions in the clinic, which were proposed to be related to the formation of protein adducts. N(α) -Boc-lysine, FEEM and IVTNTT were used as model amino acids or peptides containing one nucleophilic residue to investigate adduct types by liquid chromatography coupled with ion trap mass spectrometry (LC/MS(n) ) and high-resolution quadrupole time-of-flight mass spectrometry (Q-TOF MS). These adducts were respectively characterized as Schiff bases formed by 1:1 reaction of houttuynin with lysine or N-terminal residue and pyridinium adducts by 2:1 reaction. LC/MS(n) analysis of trypsin digests of HSA/Hb incubations with houttuynin revealed that houttuynin-modified HSA adducts were formed mainly at N-terminal amino acid and lysine residues, specifically at Lys-212, Lys-414 and Lys-525 for Schiff base adducts, and at Lys-414 and Lys-432 for pyridinium adducts, and houttuynin adducted more readily with N-terminal valine of the α- and β-chains in Hb and lysine amine (Lys-62) of the β-chain for Schiff base adducts. The results showed the direct modification of houttuynin to HSA/Hb in vitro, which was speculated to be responsible for the adverse reactions induced by houttuyniae injection.