Mapping cross-linking sites in modified proteins with mass spectrometry: An application to cross-linked hemoglobins

The combined use of trypsin digestion and peptide mass mapping by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) is reported here as an effective and rapid means for identifying the cross-linking sites in human oxy hemoglobin A (HbA) cross-linked with either bis(3,5-dibromosalicyl)succinate or -glutarate, MALDI-MS analysis of a nondigested sample of oxy HbA modified with bis(3,5-dibromosalicyl)-glutarate showed that cross-linking only occurred between the beta(1)- and beta(2)-protomers and not between alpha(1)- and alpha(2)- or alpha- and beta-protomers, along with a modification reaction on an un-cross-linked beta-chain, Results of the MALDI tryptic peptide mass maps of cross-linked hemoglobins showed several cross-linked peptides having masses consistent with: beta Val(67)-Lys(95)-XL-beta Val(67)-Lys(95), beta Val(67)-Lys(95)-XL-beta Val(67)-Arg(104), beta Val(67)-Arg(104)-XL-beta Val(67)-Arg(104) , where XL represents the succinyl or glutaryl bridging span moiety, Each of these peptides contains Lys(82), the targeted residue for these reagents, substantiating the cross-linking sites at beta(1)Lys(82)-beta(2)-Lys(82). This approach in general will enable rapid identification of the cross-linking sites in engineered proteins or intracellularly recombinant crosslinked proteins when the mass of the cross-linker and the protein primary structure are known. (C) 1996 Academic Press, Inc.