NATURAL TOXINS 5:1–13 (1997) Detection of New Spider Toxins From a Nephilengys borbonica Venom Gland Using On-Line m-Column HPLC Continuous Flow (FRIT) FAB LC/MS and MS/MS Yasuhiro Itagaki,1* Tsuyoshi Fujita, Hideo Naoki,1 Tadashi Yasuhara,2 Marta Andriantsiferana,3 and Terumi Nakajima1 1Suntory Institute for Bioorganic Research, Osaka, Japan of Nutrition Junior College of Tokyo University of Aguriculture, Tokyo, Japan 3Faculty of Science University of Antanarivo, Antanarivo, Madagascar 2Department ABSTRACT Spider venom glands store various novel neurotoxic acylpolyamines which present potent and irreversible inhibition of the glutamatergic synapses. We have developed a new highly sensitive analytical method to detect and characterize structures of those neurotoxins stored in a single venom gland using micro bore column high performance liquid chromatography (HPLC)-continuous flow FRIT fast atom bombardment mass spectrometry (HPLC-FAB/MS) and tandem mass spectrometry (MS/MS) array detection system. The high-energy collision induced dissociation (CID) spectra of sodium cationized spider toxins produced very strong structurally informative product ions which afforded the location of nitrogen atoms and the connectivity of methylene units within the polyamines. This methodology permitted detection of 40 amino acid containing acylpolyamines in the venom of Nephilengys borbonica from which structures of only five compounds were previously known. Nat. Toxins 5:1–13, 1997. r 1997 Wiley-Liss, Inc. Key Words: spider venom; acylpolyamines; liquid-chromatography/mass spectrometry; mass spectrometry/ mass spectrometry (MS/MS); charge remote fragmentation INTRODUCTION Spiders and other venomous arthropods store various complex neuroactive chemical weapons in their own venom glands which are able to paralyze and kill other arthropods. These venom toxic compounds often exhibit specific biological activities, and some of them have been used as tools in the neurochemical research. With the progress of isolation and bioassay methods in very reduced scale, spiders are receiving very close attention in the search for unique neurotoxic substances. Besides proteins and polypeptides, a series of low molecular weight glutaminergic blocker acylpolyamines, like JSTX (1–4), nephilatoxins (1–12), argiopinins (I–V), etc., were found in spider venom glands [Aramaki et al., 1987a,b; Toki et al., 1988, 1990; Skinner et al., 1990; Jasys et al., 1990; McCormick et al., 1993; Quistad et al., 1990, 1991]. For isolations and structure determinations, high performance liquid chromatography (HPLC), nuclear magnetic resonance (NMR), chemical ionization (CI), and FAB/MS are used extensively. However, these procedures require a number of venom glands and even then minor neurotoxic substances may not be detected. Since, throughout the world, more than 30,000 species of spiders from over 70 different families had been described r 1997 Wiley-Liss, Inc. by the early 1980s [McCormick and Meinwald, 1993], it is necessary to find a more efficient way to study the structures and biological activities of those neurotoxic substances, if possible by the use of a single venom gland. It has been shown that direct coupling a HPLC to MS and LC/MS/MS techniques, especially continuous flow FRIT FAB LC/MS , developed by Ito et al. [1985] and Caprioli et al. [1986] have excellent potential for handling such trace level complex mixtures. Packed m-column HPLC could be coupled with a mass spectrometer using FRIT-FAB interface without postcolumn splitting because of its low flow rate [Kassel et al., 1991a,b]. Pleasance and co-workers reported the effect of matrix additions to the mobile phase on the chromatographic separation and designed the post column FAB matrix addition system [Pleasance et al., 1990] that could maintain the polarity and viscosity of the mobile phase and permit the use of a UV detector before the introduction of samples into the mass spectrometer, thus avoiding the UV signals of the employed matrix. FAB mass spectra of spider toxins showed a characteristic (M1H)1 ion, but lacked *Correspondence to: Yasuhiro Itagaki, Suntory Institute for Bioorganic Research, Wakayamadai, Shimamoto-cho Mishima-gun Osaka, 617 Japan. Received 1 August 1996; accepted 10 October 1996. 2 ITAGAKI ET AL. Fig.1. Schematic diagram of m-column HPLC/FRIT-FAB system. The microflow cell for the UV detector was prepared from 0.32 mm i.d. fused-silica by removing polyamide coating.