Identification of amino acid residues participating in intermolecular salt bridges between self-associating proteins

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.