JOURNAL OF MASS SPECTROMETRY J. Mass Spectrom. 2004; 39: 193–201 Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jms.577 De novo sequencing of antimicrobial peptides isolated from the venom glands of the wolf spider Lycosa singoriensis B. A. Budnik, 1† J. V. Olsen, 2 T. A. Egorov, 3* V. E. Anisimova, 3 T. G. Galkina, 3 A. K. Musolyamov, 3 E. V. Grishin 3 and R. A. Zubarev 1‡ 1 Department of Chemistry, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark 2 Center for Experimental Bioinformatics, Department of Biochemistry and Molecular Biology, University of Southern Denmark, Campusvej 55, DK-5230 Odense M, Denmark 3 Laboratory of Neuroreceptors and Neuroregulators, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation Received 11 June 2003; Accepted 5 November 2003 Antimicrobial peptides (AMPs), named lycocitin 1, 2 and 3, and a peptide with a monoisotopic molecular mass of 3038.70 Da were detected in the venom glands of the wolf spider Lycosa singoriensis. Two of the peptides, lycocitin 1 and 2, are new AMPs whereas lycocitin 3 is highly homologous to lycotoxin II isolated from the venom of spider Lycosa carolinensis. In addition, two other peptides with monoisotopic masses of 2034.20 and 2340.28 Da showing the motif typical for antimicrobial peptides were also identified. These peptides and lycocitin 1, 2 and 3 were de novo sequenced using electron capture dissociation and low-energy collisional tandem mass spectrometry. The amino acid sequence of lycocitin 1 was determined as GKLQAFLAKMKEIAAQTL-NH 2 . Lycocitin 2 differs from lycocitin 1 by a replacement of a lysine residue for an arginine residue at the second position. Lycocitin 3 differs from the known lycotoxin II consisting of 27 amino acid residues by a deletion of Gly-26. Both lycocitin 1 and 2 inhibit growth of Gram-positive (Staphylococcus aureus, Bacillus subtilis) and Gram-negative (Escherichia coli) bacteria and fungi (Candida albicans, Pseudomonas aeruginosa) at micromolar concentrations. Copyright  2004 John Wiley & Sons, Ltd. KEYWORDS: antimicrobial peptides; lycocitins; Fouriers transform ion cyclotron resonance mass spectrometry; electron capture dissociation; de novo sequencing INTRODUCTION Antimicrobial peptides (AMPs) are the key elements of the innate immunity against bacteria and fungi in both the animal and plant kingdoms (for reviews, see Refs 1–6). Over 800 AMPs have already been described (http://bbcm1.univ.trieste.it/¾tossi/pag1.htm). Natural animal venoms are good sources of potential antimicrobial substances, venoms of Arthropoda being of special interest. More than 40 000 species of poisonous arthropods are cur- rently known, in which spiders and scorpions are the main species. Their venoms contain a large number of diverse biologically active components of various chemical struc- tures. However, not more than 150 arthropod species have L Correspondence to: T. A. Egorov, Laboratory of Neuroreceptors and Neuroregulators, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation. E-mail: ego@ibch.ru † Present address: Cardiovascular Proteomics Center, School of Medicine, Boston University, Boston, Massachusetts 02118–2543, USA. ‡ Present address: Biological and Medical Research Laboratory, Uppsala University, S-75121 Uppsala, Sweden. been investigated so far and only few of them have been characterized for the presence of antimicrobial activity. Two types of linear cationic AMPs consisting of 13–27 amino acid residues were found in arthropod venoms: cecropin- like and mellitin-like types. 7 The AMPs of the first type are mainly detected in the hemolymph of various arthropods such as sacrotoxin in house fly, 8 cecropins in lepidoptera 9 and spinegirin in termites. 10 AMPs of the second type were found in the venom glands of bees, 11 wasps, 12 ants, 13 scorpions 14 and spiders such as Lycosa carolinensis, 15 Cupi- enius salei 16 and Oxyopes kitabensis. 17 The composition of tarantula spider venom in relation to sex, age and geograph- ical origin was studied by Escoubas and co-authors using high-performance liquid chromatography (HPLC) and cap- illary electrophoresis in combination with different mass spectrometric methods. 18 – 21 In this paper, we describe the isolation, structural evaluation and biological activity of lycocitin 1, 2 and 3 and two peptides that are possible candidates for AMPs from the venom glands of the wolf spider Lycosa singoriensis. Copyright  2004 John Wiley & Sons, Ltd.