UV-MALDI-MS fingerprints for monitoring biosystems: the case of toads secretions

PETROSELLI, GABRIELA; ERRA BALSELLS ROSA; NONAMI HIROSHI

Osaka

Congreso; Mass Spectrometry and Proteomics 2018; 2018

The Mass spectrometry Society of Japan

In many amphibians, the granular glands can be grouped in special regions forming macroglands. This is the case of toads, characterized by the presence of a pair of parotoid macroglands, strategically located to give protection by poison release in case of attacks. Common toads are divided in genera Rhinella and Rhaebo in South America (used to be grouped in genus Bufo).1 Rhinella arenarum is a native species from South America and is widely distributed in Argentina.2 Anurans have developed different resources to protect themselves against predators and microbial infection, which can include ecological, morphological, physiological or behavioural features. Chemical defenses represent a common strategy in amphibians. The number and diversity of compounds produced by amphibians in their skin glands is surprisingly high. Those secretions contain biogenic amines (i.e., adrenaline and noradrenaline), steroids (bufogenines and bufotoxins), alkaloids (i.e., serotonin, batrachotoxin and tetrodotoxin) and peptides (including smaller oligopeptides, polypeptides and proteins). The abundance and diversity of components of each of these family?s compounds can vary according to the life history of the amphibian in question, gender and season. Chemical composition of secretion produced by Rhinella arenarum was studied for the first time in 1933 and the presence of arenobufagin, arenobufotoxin and arenobufotenin in the alcoholic extract was found.3 Later, some bufogenins using IR spectroscopy in paratoid secretions were described.4 More recently, Maciel et al. (2003) studied indolealkylamines by TLC and ESI-MS in extract from secretions.5 Moreover, Mebs et al.(2007) using skin, instead of secretion, described the presence of bufogenine and bufalin by HPLC-ESI.6Additionally, several studies identified compounds in secretion from other species such as Rhinella genus and Rhaebo genus found in Brazil. It was described significant differences in composition between R. marina and R. guttatus venoms but no relevant chemical difference were observed between male and female secretions. Combination of techniques such as chromatographic isolation, NMR and MS; reversed phase HPLC and MALDI MS/MS. nano UPLC-ESI allowed characterization of poison components of different Rhinella species. In the secretions under study they detected several bufadienolides (i.e., dehydrobufotenine, hellebrigenin, telocinobufagin, serotonin, N´-methyl-serotonin, resinobufagin, hellebrigenol-3-O-sulfate, bufalin, N´-N´-dimethyl-serotonin (bufotenin), desacetylcinobufagin and marinobufagin). Although some components were common among the secretions, several molecules were identified as exclusive to some species. It was concluded that biological features, and not only evolution, seem to directly influence the skin secretion composition.In this work, using Rhinella arenarum we have performed, for the first time, the MALDI-MS and MS/MS characterization of the components of the secretion used as crude material, just suspended in MeOH (or MeCN). Samples were collected from juvenile and adult specimens (non-reproductive the former and reproductive the latter, respectively) at two different times in the year, July (not reproductive season) and December (reproductive season). The crude sample as a whole (whole suspension), was spotted on the MALDI plate for analysis. ESI-Orbitrap was used for cross-checking experiments. Thus, for the first time both MS techniques were used on this crude material to identify distinctive bufadienolide composition profiles (fingerprints), for male and female. The pattern of signals obtained at m/z ranges 600-800 and 1200-1600 could be assigned as the argininyl bufadienolide esters fingerprint characteristic of female and male. Interesting differences in fingerprint and Na+ contents were observed depending on gender.Those findings showed that MALDI-MS fingerprints from crude (raw) secretions can be a tool of choice for characterization of amphibian?s secretions and for finding out new biomolecules naturally present in the material but may be lost during chemical manipulation (work-up) of the secretions.