Poster 188 - Maldi Tof mass spectrometry and de novo sequence as a tool to investigate and identify

Poster 188. Congresso da Sociedade Brasileira de Toxinologia, 8., Symposium of the Pan American Section of the International Society on Toxinology, 8., 2004, Angra dos Reis, Brasil. Abstracts... J. Venom. Anim. Toxins incl.Trop. Dis., 2004, 10, 3, p.547.

Maldi Tof Mass Spectrometry and de novo Sequence as a Tool to Investigate and Identify Low Mass Peptides in Snake Venoms

Wermelinger, L.S.¹; Dutra, D.L.S.¹; Oliveira-Carvalho, A.L.¹; Soares, M.S.²; Bloch Jr.,C.³; and Zingali, R.B.¹

1 Dep. Bioq. Medica, ICB, CCS, UFRJ. RJ, 2 IBCCF, CCS, UFRJ. Rede Proteômica do Rio de Janeiro, ³ LNLS, Campinas-SP Brasil.

Characterization of the peptide content in snake venoms can be an important tool to investigate new pharmacological lead compounds. In order to analyze with detail all peptides and small proteins present in crude samples, technologies other than 2-D gel electrophoresis are required. One important application of MALDI TOF/MS is direct analysis of complex samples allowing identification of low mass peptides and small proteins in a single step. In fact, this tool has been successfully used for the identification of a number of peptides in crude venoms. At this point, reproducible profiles for MS peaks may reveal important similarities and discrepancies among venoms from distinct species. The aim of this work is to use the MALDI-TOF/MS approach to obtain a mass pattern of the major peptides (<10 kDa) present in different pooled venoms from subfamilies. We also used MALDI TOF/TOF to obtain MS/MS sequence of the peptides. Viperinae and Crotalinae venoms from four different Bothrops sp. (B. jararaca, B. insularis, B. jararacussu, and B. neuwiedi), and three Crotalus sp. (C. viridis, C. adamanteus and C. durissus terrficus) were analyzed. MALDI-TOF mass spectra was obtained in the range 800-10.000 m/z, using CHCA and SA as matrix in the linear mode. Peptide de novo sequencing was preformed by precursor ion fragmentation using N2 and/or air collision to induce dissociation. Collision cell pressure was kept at 2.8 X 10 –6 torr. In agreement with previous reports, all venoms from Viperinae subfamilies showed a great amount of peptides in the range of 1000-2000 and 6000-8000 m/z, possible due to the presence of bradykinin-potentiating peptides and disintegrins. The de novo sequencing of some peptides ranging from 1000 to 2000 of the bothropic venoms confirmed the presence of BPPs peptides. Venoms from Crotalinae subfamily additionally showed peptides in mass in the range of 4000-5000 m/z in accordance with the presence of crotamin isoforms. In order to verify if additional peptides could be seen after partial purification, B. insularis venom was submitted to gel-filtration chromatography. Two pools of low molecular weight were analyzed by MALDI-TOF. No additional peptides were detected, indicating that the main peptides from crude venoms can be observed without pre-purification steps.