title: Identification of amino acid residues participating in intermolecular salt bridges between self-associating proteins.
abstract: Salt bridges between self-associating hen egg white (HEW) lysozyme and bovine insulin molecules were converted to covalent links by ethanedinitrile (cyanogen) and identified using mass spectrometry. Peptides resulting from cyanogen-mediated intermolecular cross-linking of HEW lysozyme were detected using in-gel digestion and matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Sequence data from electrospray ionization-quadrupole-time of flight mass spectrometry (ESI-Q-TOF MS) revealed that one of the peptides has a covalent bond between Asp 66 and Arg 14. The self-assembly of bovine insulin was also investigated using cyanogen. Using high-performance liquid chromatography (HPLC) coupled with ESI-Q-TOF MS, an intermolecular salt bridge association was identified by covalently linking the B chain C-terminal carboxyl group of Ala 30 and the charged imidazole of His 5 (B chain). A method was developed incorporating cyanogen, enzymatic digestion, one-dimensional gel electrophoresis, MALDI-TOF MS, and HPLC ESI-Q-TOF MS to identify amino acid residues participating in salt bridge formations at protein-protein interfaces. The novelty of this approach is the ease with which cyanogen can be administered to a protein sample and the apparent lack of nonspecific cross-linking side reactions interfering with the analysis.
authors.affiliation: Department of Chemistry, University of Cincinnati, Cincinnati, OH 45221-0172, USA.
authors.forename: Michael S
authors.initials: MS
authors.lastname: Winters
authors.forename: Richard A
authors.initials: RA
authors.lastname: Day
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis / Q000737:chemistry*; D000818:Animals; D002417:Cattle; D002645:Chickens; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003432:Cross-Linking Reagents / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel; D007328:Insulin / Q000737:chemistry*; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry*; D009570:Nitriles / Q000737:chemistry*; D010063:Ovum / Q000201:enzymology; D010446:Peptide Fragments / Q000737:chemistry; D017510:Protein Folding; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D055672:Static Electricity; D014357:Trypsin / Q000737:chemistry
chemical_list: D000596:Amino Acids; D003432:Cross-Linking Reagents; D007328:Insulin; D009570:Nitriles; D010446:Peptide Fragments; C011206:cyanogen; D009113:Muramidase; D014357:Trypsin