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 (K_d > 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 option to perform mass spectrometric peptide sequencing by so-called post-source decay (PSD) product ion analysis, (even with subpicomoles of analyte loads in heterogeneous samples) allowed us not only to determine the labeled tryptic peptide but also to localize the site of the crosslink down to the A268 position of the delta-subunit. It is felt that these results demonstrate the capability of the MALDI technique to overcome some of the constraints inherent in the analytical tool of photolabeling, especially in cases where large molecular probes (such as NT II) are employed.