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

G. PETROSELLI; R.; ERRA BALSELLS; H. NONAMI

Osaka

Conferencia; Spectrometry and Proteomics 2018 (MSP2018); 2018

Japan Society Mass Spectrometry

UV-MALDI-MS fingerprints formonitoring biosystems: the case of toads secretions(Universityof Buenos Aires1, Ehime University2)G.Petroselli1, ??R. Erra-Balsells1,H. Nonami2  Keywords: Rhinellaarenarum; paratoid secretion; bufadienolides.    Inmany amphibians, the granular glands can be grouped in special regions formingmacroglands. This is the case of toads, characterized by the presence of a pairof parotoid macroglands, strategically located to give protection by poisonrelease 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 iswidely distributed in Argentina.2 Anurans have developed differentresources to protect themselves against predators and microbial infection, whichcan include ecological, morphological, physiological or behavioural features.Chemical defenses represent a common strategy in amphibians. The number anddiversity of compounds produced by amphibians in their skin glands issurprisingly high. Those secretions contain biogenic amines (i.e., adrenalineand noradrenaline), steroids (bufogenines and bufotoxins), alkaloids (i.e.,serotonin, batrachotoxin and tetrodotoxin) and peptides (including smalleroligopeptides, polypeptides and proteins). The abundance anddiversity of components of each of these family?fs compounds can vary accordingto the life history of the amphibian in question, gender and season.   Chemical composition of secretion produced byRhinella arenarum was studied for thefirst time in 1933 and the presence of arenobufagin, arenobufotoxin andarenobufotenin in the alcoholic extract was found.3 Later, somebufogenins using IR spectroscopy in paratoid secretions were described.4More recently, Maciel et al. (2003) studied indolealkylamines by TLC and ESI-MSin 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 identifiedcompounds in secretion from other species such as Rhinella genus and Rhaebogenus found in Brazil. It was described significant differences in compositionbetween R. marina and R. guttatus venoms but no relevantchemical 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 ofpoison components of different Rhinella species. In the secretions under studythey detected several bufadienolides (i.e., dehydrobufotenine, hellebrigenin,telocinobufagin, serotonin, N?L-methyl-serotonin, resinobufagin,hellebrigenol-3-O-sulfate, bufalin, N?L-N?L-dimethyl-serotonin (bufotenin),desacetylcinobufagin and marinobufagin). Although some components were commonamong the secretions, several molecules were identified as exclusive to somespecies. It was concluded that biological features, and not only evolution,seem to directly influence the skin secretion composition.  Inthis work, using Rhinella arenarum wehave performed, for the first time, the MALDI-MS and MS/MS characterization ofthe components of the secretion used as crude material, just suspended in MeOH(or MeCN). Samples were collected fromjuvenile and adult specimens (non-reproductive the former and reproductive thelatter, respectively) at two different times in the year, July (notreproductive season) and December (reproductive season). The crude sampleas 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 usedon this crude material to identify distinctive bufadienolide compositionprofiles (fingerprints), for male and female. The pattern of signals obtainedat m/z ranges 600-800 and 1200-1600 could be assigned as the argininylbufadienolide esters fingerprint characteristic of female and male. Interesting differences in fingerprint and Na+contents were observed depending on gender.  Thosefindings showed that MALDI-MS fingerprints from crude (raw) secretions can be atool of choice for characterization of amphibian?fs secretions and for findingout new biomolecules naturally present in  the material but may be  lost during chemical manipulation (work-up)of the secretions. ReferencesD.R. Frost,Amphibian Species of the World http://research.amnh.org/herpetology/amphibia/index.html (2016)J.M. Gallardo, Neotropica 31 23- 28 (1964)K.K. Chen et al., JPET 49 1-13 (1933) R. Rees et al., Helvetica 42 2400-2418 (1959)N.M. Maciel et al., CompBiochem Physiol B Bioche Mol Biol. 134 641-649 (2003)D. Mebs et al., Comp Biochem Physiol C Toxicol Pharmacol.144 398-402 (2007)