Structural determination of the conjugate of human serum albumin with a mitomycin C derivative, KW-2149, by matrix-assisted laser desorption/ionization mass spectrometry

new mitomycin C derivative, KW-2149, is known to form a covalent conjugate with human serum albumin (HSA). This conjugate exhibits 1/20 of the anticellular activity of unconjugated KW-2149. Structural studies of this conjugate were carried out using a combination of enzymatic digestion, high-performance liquid chromatography (HPLC), and matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The tryptic peptide T5 (residues 21-41) was the only peptide found to be modified by KW-2149 moieties, the [(gamma-L-glutamylamino)ethyl]thio group or the (2-aminoethyl)thio group, through a disulfide bond. Although the latter peptide lost its mitomycin C moiety in the course of tryptic digestion, these data strongly suggest that KW-2149 was bound to Cys-34, the only free cysteine on KSA.