Mass Spectrometric Structure Determination of Spider Toxins: Arginine-Containing Acylpolyamines From Venoms of Brazilian Garden Spider Nephilengys cruentata Mario Sergio Palma, 2 Yasuhiro Itagaki, 1 * Tsuyoshi Fujita, 1 Miki Hisada, 1 Hideo Naoki, 1 and Terumi Nakajima 1 1 Sunto ry Institute fo r Bio o rganic Re se arc h, O saka, Jap an 2 Lab o rato ry o f Mo le c ular Bio lo gy, Institute o f Bio sc ie nc e s/CEVAP , Unive rsity o f Sa ˜o Paulo State (UNESP), Sao Paulo , Braz il ABSTRACT A ne w strate gy to c harac te riz e glutamine rgic b lo c ke r ac ylp o lyamine s sto re d in a spider ve no m w ith mass sp e c tro me try is d e sc rib e d . The c rud e sp id e r ve no m e xtrac ts are ame nab le to d ire c t MA LDI mass sp e c tro me try analysis w hic h p ro vid e s a rap id and ac c urate me ans o f me asuring the mo le c ular w e ights o f ac ylp o lyamine s w itho ut the iso latio n o f ind ivid ual samp le s. Co mp are d w ith the p re vio usly d e ve lo p e d m-c o lumn HPLC/MS me tho d , this p ro c e d ure p ro vid e s mo re e ffic ie nt d e te c tio n and id e ntific atio n o f c o mp le x ve no m c o nstitue nts. Tw e nty-five ac ylp o lyamine s w e re d e te c te d fro m Braz ilian gard e n sp id e r Ne p hile ngys c rue ntata c rud e ve no m e xtrac ts b y b o th HPLC/MS and MA LDI-mass sp e c tro me try. The se ac ylp o lyamine struc ture s w e re d e te rmine d b y high-e ne rgy c o llisio n ind uc e d d isso c iatio n MS/MS me tho d . Mo st o f the c o mp o und s w e re c lassifie d into the p re vio usly re p o rte d ge ne raliz e d struc ture s typ e s A to D, w hic h w e re fo und fro m the ve no m o f Ne p hile ngys b o rb o nic a. The struc ture s o f fo ur ac ylp o lyamine s (M 1 H) 1 , m/z 623, 646, 688, and 745, w hic h w e re no t c o ntaine d in the ve no m o f Ne p hile ngys b o rb o nic are w e re d e te rmine d to have arginine at the p o lyamine c hain te rminal and w e re name d NPTX-622, -645, -687, and -744, re sp e c tive ly. Nat. To xins 5:47–57, 1997. r 1997 Wile y-Liss, Inc . Key Words: spider venom; ac ylp o lyamine s; matrix-assiste d lase r d e so rp tio n io niz atio n (MALDI)-mass sp e c tro me try; high-e ne rgy c o llisio n ind uc e d d isso c iatio n (CID) INTRO DUCTIO N Venomous animals store various complex substances which often exhibit specific biological activities, and some of them have been used as tools in neurochemical research. But full characterization of the venom constituents was still difficult because of structurally related complex mixtures [McCormick and Meinwald, 1993]. Recently, with the progress of isolation and bioassay methods, spiders are receiving high attention in the search for new neurotoxic substances and have yielded a series of low molecular weight glutaminergic blocker acylpolyamines [Aramaki et al., 1987a,b; Adams et al., 1987; Toki et al., 1988; Budd et al., 1988; Grishin et al., 1989; Jasys et al., 1990; Quistad et al., 1990; Chiba et al., 1994,1995]. We have also reported a highly sensitive analytical method to characterize complex neurotoxic compounds extracted from spider venoms using fast-atom bombardment (FAB) and four-sector tandem mass spectrometry (MS/MS) [Nakajima and Itagaki, 1996; Itagaki et al., 1996]. With the use of μ-column HPLC/FAB-MS system, rapid identification of nearly every acylpolyamine in a crude venom extract is permitted. From the crude venom extracts of Nephilengys borbonica, it was possible to identify 40 acylpolyamines, and among those only five compounds were previously known [Itagaki et al., 1996]. When compared with the traditional procedures for elucidat- ing the structures of spider toxins which have been the use of high performance liquid chromatography to collect biologi- cally active fractions, followed by proton nuclear magnetic resonance measurements and/or hydrolysis to amino acids and amines, this μ-column HPLC/FAB-MS system can be seen as an advanced method because all venom constituents regardless of being major or minor substances can be detected at the same time with high analytical accuracy and less sample. But still, the use of μ-column LC requires time and laborious efforts to obtain analytical results. Matrix- assisted laser desorption ionization (MALDI) mass spectrom- etry has emerged as an important technique for the analysis of biological complex mixtures without prior isolation *Co rre sp o nd e nc e to : Yasuhiro Itagaki, Sunto ry Institute fo r Bio o rganic Re se arc h, Wakayamad ai, Shimamo to -c ho Mishima-gun O saka, 618 Jap an. Received 23 October1996; Accepted 13 December1996 NATURAL TOXINS 5:47–57 (1997) r 1997 Wile y-Liss, Inc .