Spider venoms are composed of hundreds of proteins and peptides. Several of these venom toxins are cysteine-rich peptides in the mass range of 3–9 kDa. Small peptides (<3 kDa) can be fully characterized by mass spectrometry analysis, while proteins are generally identified by the bottom-up approach in which proteins are first digested with trypsin to generate shorter peptides for MS/MS characterization. In general, it is sufficient for protein identification to sequence two or more peptides, but for venom peptidomics it is desirable to completely elucidate peptide sequences and the number of disulfide bonds in the molecules. In this chapter we describe a methodology to completely sequence and determine the number of disulfide bonds of spider venom peptides in the mass range of 3–9 kDa by multiple enzyme digestion, mass spectrometry of native and digested peptides, de novo analysis, and sequence overlap alignment.
Spider venom Peptidomics Mass spectrometry De novo analysis
This is a preview of subscription content, log in to check access.
Springer Nature is developing a new tool to find and evaluate Protocols. Learn more
This work was supported by grants from Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP, 2012/19321-9 and 2016/03839-0 to A. K. T.), Financiadora de Estudos e Projetos (FINEP) and Master’s degree fellowships from FAPESP (2014/17140-2) to R. L. L., and from Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) to E. S. N.
Escoubas P, Sollod B, King GF (2006) Venom landscapes: mining the complexity of spider venoms via a combined cDNA and mass spectrometric approach. Toxicon 47:650–663CrossRefPubMedGoogle Scholar
Palagi A, Koh JMS, Leblanc M et al (2013) Unravelling the complex venom landscapes of lethal Australian funnel-web spiders (Hexathelidae: Atracinae) using LC-MALDI-TOF mass spectrometry. J Proteome 80:292–310CrossRefGoogle Scholar
Abreu TF, Sumitomo BN, Nishiyama MY et al (2017) Peptidomics of Acanthoscurria gomesiana spider venom reveals new toxins with potential antimicrobial activity. J Proteome 151:232–242CrossRefGoogle Scholar
Norton RS, Pallaghy PK (1998) The cystine knot structure of ion channel toxins and related polypeptides. Toxicon 36:1573–1583CrossRefPubMedGoogle Scholar
Wang X, Connor M, Smith R et al (2000) Discovery and characterization of a family of insecticidal neurotoxins with a rare vicinal disulfide bridge. Nat Struct Biol 7:505–513CrossRefPubMedGoogle Scholar
Peng K, Lin Y, Liang SP (2006) Nuclear magnetic resonance studies on Huwentoxin-XI from the Chinese bird spider Ornithoctonus huwena: 15N labeling and sequence-specific 1H, 15N nuclear magnetic resonance assignments. Acta Biochim Biophys Sin Shanghai 38:457–466CrossRefPubMedGoogle Scholar
Rates B, Prates MV, Verano-Braga T et al (2013) μ-Theraphotoxin-An1a: primary structure determination and assessment of the pharmacological activity of a promiscuous anti-insect toxin from the venom of the tarantula Acanthoscurria natalensis (Mygalomorphae, Theraphosidae). Toxicon 70:123–134CrossRefPubMedGoogle Scholar
Wan H, Lee KS, Kim BY et al (2013) A spider-derived Kunitz-type serine protease inhibitor that acts as a plasmin inhibitor and an elastase inhibitor. PLoS One 8:e53343CrossRefPubMedPubMedCentralGoogle Scholar
Ayroza G, Ferreira ILC, Sayegh RSR et al (2012) Juruin: an antifungal peptide from the venom of the Amazonian Pink Toe spider, Avicularia juruensis, which contains the inhibitory cystine knot motif. Front Microbiol 3:324CrossRefPubMedPubMedCentralGoogle Scholar
Windley MJ, Herzig V, Dziemborowicz SA et al (2012) Spider-venom peptides as bioinsecticides. Toxins (Basel) 4:191–227CrossRefGoogle Scholar
Mourão CB, Heghinian MD, Barbosa EA et al (2013) Characterization of a novel peptide toxin from Acanthoscurria paulensis spider venom: a distinct cysteine assignment to the HWTX-II family. Biochemistry 52:2440–2452CrossRefPubMedGoogle Scholar
Tashima AK, Zelanis A, Kitano ES et al (2012) Peptidomics of three bothrops snake venoms: insights into the molecular diversification of proteomes and peptidomes. Mol Cell Proteomics 11:1245–1262CrossRefPubMedPubMedCentralGoogle Scholar
Sasaki K, Osaki T, Minamino N (2013) Large-scale identification of endogenous secretory peptides using electron transfer dissociation mass spectrometry. Mol Cell Proteomics 12:700–709CrossRefPubMedGoogle Scholar
Rocha-e-Silva TAA, Sutti R, Hyslop S (2009) Milking and partial characterization of venom from the Brazilian spider Vitalius dubius (Theraphosidae). Toxicon 53:153–161CrossRefPubMedGoogle Scholar