Probing the non-covalent binding interaction of the Na+ channel inactivation gate peptide in a linker between domain III and IV with 5,5-diphenyhydantoin in electrospray ion trap tandem mass spectrometry.
The Na(+) channel-subunit containing an Ile1488, Phe1489 and Met1490 (IFM) motif is critical for a fast inactivation process. BL-1, a model IFM-containing peptide with a sequence of acetyl-GGQDIFMTEEK-OH, was observed as a doubly charged potassium-adduct ion by electrospray ionization mass spectrometry (ESI-MS) and a singly charged ion by atmospheric-pressure matrix-assisted laser desorption/ionization mass spectrometry (AP-MALDI-MS). Two crown ethers were applied to demonstrate their desalting ability and then to confirm the potassium-adduct assignments. In order to probe the best binding condition for BL-1 with a local anesthetic drug, 5,5-diphenyhydantoin (DPH), a series of experiments were performed and the parameters affecting complexation were carefully investigated including molar ratios, reaction time and reaction temperature. The most effective conditions for the observation of the complex by ESI-MS were molar ratio of BL-1 and DPH of 1:28 after 18 h of incubation at 40 degrees C. In addition, collision-activated dissociation (CAD) was successfully applied to confirm the formation of the complex between BL-1 with DPH that is via a weak non-covalent bonding with a 1:1 stoichiometry.
Lou
Bih-Show
BS
Center of General Education, Chang Gung University, Tao-Yuan 333, Taiwan.
Chen
Yuan-Chin
YC
Wu
Hui-Fen
HF
D015640:Ion Channel Gating; D015336:Molecular Probe Techniques; D010455:Peptides / Q000737:chemistry*; D010672:Phenytoin / Q000737:chemistry*; D017434:Protein Structure, Tertiary; D015222:Sodium Channels / Q000737:chemistry*; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010455:Peptides; D015222:Sodium Channels; D010672:Phenytoin