Matrix-assisted laser desorption ionization (MALDI) and post source decay (PSD) product ion mass analysis localize a photolabel crosslinked to the delta-subunit of nAChR protein by neurotoxin II

Abstract

In an attempt to probe the molecular topology of the ion gate in the nicotinic AChR protein, the high affinity (Kd > 10−11 molar) ligand neurotoxin II (NT II), a 61 residue peptide from the venom of Naja naja oxiana, was photocrosslinked to the nAChR of Torpedo californica. The photoactivatable group was a (125I)-p-azidosalicylamidoethyl-1,3-dithiopropyl (ASED) moiety located at the K25 residue of NT II. Specific labeling occurred at the delta-subunit which, after SDS-PAGE, was subjected to tryptic digestion and reverse phase high performance liquid chromatography (rpHPLC) separation. Although the radioactivity profile of the collected HPLC fractions exhibited two sharp maxima suggesting rather specific crosslinking site(s), attempts to localize these sites by Edman degradation failed. Thus, matrix-assisted laser desorption ionization (MALDI) mass spectrometry was employed in the hope of circumventing the difficulties encountered with the conventional protocols. This technique, in conjunction with the opt...