title: Mapping cross-linking sites in modified proteins with mass spectrometry: an application to cross-linked hemoglobins.
abstract: 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 Val67-Lys95-XL-beta Val67-Lys95, beta Val67-Lys95-XL-beta Val67-Arg104, beta Val67-Arg104-XL-beta Val67-Arg104, where XL represents the succinyl or glutaryl bridging span moiety. Each of these peptides contains Lys82, the targeted residue for these reagents, substantiating the cross-linking sites at beta 1Lys82-beta 2Lys82. This approach in general will enable rapid identification of the cross-linking sites in engineered proteins or intracellularly recombinant cross-linked proteins when the mass of the cross-linker and the protein primary structure are known.
authors.affiliation: Department of Chemistry, University of Wisconsin-Eau Claire 54702, USA. yangt@uwec.edu
authors.forename: T
authors.initials: T
authors.lastname: Yang
authors.forename: D R
authors.initials: DR
authors.lastname: Horejsh
authors.forename: K J
authors.initials: KJ
authors.lastname: Mahan
authors.forename: E J
authors.initials: EJ
authors.lastname: Zaluzec
authors.forename: T J
authors.initials: TJ
authors.lastname: Watson
authors.forename: D A
authors.initials: DA
authors.lastname: Gage
mesh_terms: D000595:Amino Acid Sequence; D001241:Aspirin / Q000031:analogs & derivatives / Q000737:chemistry; D001665:Binding Sites; D003432:Cross-Linking Reagents / Q000737:chemistry*; D006441:Hemoglobin A / Q000737:chemistry*; D006801:Humans; D008969:Molecular Sequence Data; D010108:Oxyhemoglobins / Q000032:analysis / Q000737:chemistry*; D010455:Peptides / Q000032:analysis / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014357:Trypsin
chemical_list: D003432:Cross-Linking Reagents; D010108:Oxyhemoglobins; D010455:Peptides; C021631:bis(3,5-dibromosalicyl)succinate; C036858:bis(3,5-dibromosalicyl)glutarate; D006441:Hemoglobin A; D014357:Trypsin; D001241:Aspirin