Biochemical Analyses of Proteins from Duttaphrynus melanostictus (Bufo melanostictus) Skin Secretion: Soluble Protein Retrieval from a Viscous Matrix by Ion-Exchange Batch Sample Preparation
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A crucial step in scientific analysis can be sample preparation, and its importance increases in the same rate as the sensitivity of the following employed/desired analytical technique does. The need to analyze complex, viscous matrices is not new, and diverse approaches have been employed, with different success rates depending on the intended molecules. Solid-phase extraction, for example, has been successfully used in sample preparation for organic molecules and peptides. However, due to the usual methodological conditions, biologically active proteins are not successfully retrieved by this technique, resulting in a low rate of protein identification reported for the viscous amphibian skin secretion. Here we describe an ion-exchange batch processing sample preparation technique that allows viscous or adhesive materials (as some amphibian skin secretions) to be further processed by classical liquid chromatography approaches. According to our protocol, samples were allowed to equilibrate with a specific resin that was washed with appropriated buffers in order to yield the soluble protein fraction. In order to show the efficiency of our methodology, we have compared our results to classically prepared skin secretion, i.e., by means of filtration and centrifugation. After batch sample preparation, we were able to obtain reproductive resolved protein chromatographic profiles, as revealed by SDS-PAGE, and retrieve some biological activities, namely, hydrolases belonging to serine peptidase family. Not only that, but also the unbound fraction was rich in low molecular mass molecules, such as alkaloids and steroids, making this sample preparation technique also suitable for the enrichment of such molecules.
KeywordsBatch chromatography Ion-exchange chromatography Viscous sample Amphibian skin secretion Hydrolase
We are thankful to Dr. Ivo Lebrun for the thoughtful considerations about the batch sample preparation and for providing the IEX media.
This work was funded by CAPES (DOCM, 969130 Grant), FINEP (Financiadora de Estudos e Projetos) (Grant Number 01.09.0278.04), FAPESP and CNPq. DCP is a CNPq fellow researcher (Conselho Nacional de Desenvolvimento Científico e Tecnológico) (Grant 303792/2016-7).
Compliance with Ethical Standards
Conflict of interest
Authors declare that they have no conflict of interest.
This article does not contain any studies with animals performed by any of the authors.
- 8.Mariano DOC, Yamaguchi LF, Jared C, Antoniazzi MM, Sciani JM, Kato MJ, Pimenta DC (2015) Pipa carvalhoi skin secretion profiling: absence of peptides and identification of kynurenic acid as the major constitutive component. Comp Biochem Physiol 167:1–6Google Scholar
- 9.Sousa-Filho LM, Freitas CD, Lobo MD, Monteiro-Moreira AC, Silva RO, Santana LA, Ribeiro RA, Souza MH, Ferreira GP, Pereira AC, Barbosa AL, Lima MS, Oliveira JS (2016) Biochemical profile, biological activities, and toxic effects of proteins in the Rhinella schneideri parotoid gland secretion. J Exp Zool A 325:511–523CrossRefGoogle Scholar
- 11.Frost DR (2017) Amphibian species of the world: an online reference. Version 6.0. American Museum of Natural History, New York. http://research.amnh.org/herpetology/amphibia/index.html. Accessed 01 Feb 2017
- 14.Conceição K, Bruni FM, Sciani JM, Konno K, Melo RL, Antoniazzi MM, Jared C, Lopes-Ferreira M, Pimenta DC (2009) Identification of bradykinin: related peptides from Phyllomedusa nordestina skin secretion using electrospray ionization tandem mass spectrometry after a single-step liquid chromatography. J Venom Anim Toxins Incl Trop Dis 15:633–652CrossRefGoogle Scholar
- 17.Sousa LQ, Machado KD, Oliveira SF, Araújo LD, Monção-Filho ED, Melo-Cavalcante AA, Vieira-Júnior GM, Ferreira PM (2017) Bufadienolides from amphibians: a promising source of anticancer prototypes for radical innovation, apoptosis triggering and Na+/K+-ATPase inhibition. Toxicon 127:63–76CrossRefPubMedGoogle Scholar
- 20.König E, Wesse C, Murphy AC, Zhou M, Wang L, Chen T, Shaw C, Bininda-Emonds OR (2013) Molecular cloning of the trypsin inhibitor from the skin secretion of the Madagascan Tomato Frog, Dyscophus guineti (Microhylidae), and insights into its potential defensive role. Org Divers Evol 13:453–461CrossRefGoogle Scholar
- 33.Conceição K, Konno K, de Melo RL, Antoniazzi MM, Jared C, Sciani JM, Conceição IM, Prezoto BC, de Camargo AC, Pimenta DC (2007) Isolation and characterization of a novel bradykinin potentiating peptide (BPP) from the skin secretion of Phyllomedusa hypochondrialis. Peptides 28:515–523CrossRefPubMedGoogle Scholar
- 35.GE Healthcare (2010) Strategies for protein purification: handbook. GE Healthcare, UppsalaGoogle Scholar
- 38.Rawlings ND, Barrett AJ (2013) Handbook of proteolytic enzymes. In: Introduction: serine peptidases and their clans. Academic Press, New York, pp 2491–2523Google Scholar
- 45.Zhao Y, Jin Y, Lee WH, Zhang Y (2006) Purification of a lysozyme from skin secretions of Bufo andrewsi. Comp Biochem Physiol C 142:46–52Google Scholar