Fast analysis of low molecular mass compounds present in snake venom: identification of ten new...

Characterization of the peptide content in snake venoms can be an important tool for the investigation of new pharmacological lead compounds. For this purpose, single-step analysis of crude venoms has recently been demonstrated using mass spectrometry (MS) techniques. Reproducible profiles of ions in MS and MS/MS spectra may also be used to compare venoms from different species. In this work matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS) was used to obtain mass patterns of the major peptides (<8 kDa) found in pooled venoms from the genera Bothrops and Crotalus. Venoms from five different Bothrops species (B. jararaca, B. insularis, B. alternatus, B. jararacussu, and B. neuwiedi) and three Crotalus species (C. viridis, C. adamanteus and C. durissus terrificus) were analyzed. In agreement with other reports, venoms from Bothrops species contained a variety of peptides in the range m/z 1000-1500, and in some samples larger components (m/z 7000-8000) were detected. In the Crotalus species venoms were rich in peptides ranging from m/z 1000-1500 and 4000-5500. MS/MS experiments on the low molecular mass peptides (m/z 1000-1500) confirmed the presence of ten new bradykinin-potentiating peptides among venoms from genera Bothrops and Crotalus. In order to determine whether additional peptides could be identified after partial purification, B. jararaca venom was subjected to size-exclusion chromatography on Sephacryl S-200, and two distinct low molecular mass pools were analyzed further by MALDI-TOFMS. No additional peptides were detected from the pool with masses below 2000 Da but a substantial improvement with better resolution was observed for the pool with masses above 7000 Da, indicating that complex samples such as crude snake venoms can be analyzed for low molecular mass peptides using a single-step procedure.