The Protein Journal

, Volume 36, Issue 4, pp 308–321 | Cite as

A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis

  • Fernando Lledías
  • Felipe Hernández
  • Viridiana Rivas
  • Abisaí García-Mendoza
  • Gladys I. Cassab
  • Jorge Nieto-Sotelo
Article

Abstract

Crassulacean acid metabolism plants have some morphological features, such as succulent and reduced leaves, thick cuticles, and sunken stomata that help them prevent excessive water loss and irradiation. As molecular constituents of these morphological adaptations to xeric environments, succulent plants produce a set of specific compounds such as complex polysaccharides, pigments, waxes, and terpenoids, to name a few, in addition to uncharacterized proteases. Since all these compounds interfere with the analysis of proteins by electrophoretic techniques, preparation of high quality samples from these sources represents a real challenge. The absence of adequate protocols for protein extraction has restrained the study of this class of plants at the molecular level. Here, we present a rapid and reliable protocol that could be accomplished in 1 h and applied to a broad range of plants with reproducible results. We were able to obtain well-resolved SDS/PAGE protein patterns in extracts from different members of the subfamilies Agavoideae (Agave, Yucca, Manfreda, and Furcraea), Nolinoideae (Dasylirion and Beucarnea), and the Cactaceae family. This method is based on the differential solubility of contaminants and proteins in the presence of acetone and pH-altered solutions. We speculate about the role of saponins and high molecular weight carbohydrates to produce electrophoretic-compatible samples. A modification of the basic protocol allowed the analysis of samples by bidimensional electrophoresis (2DE) for proteomic analysis. Furostanol glycoside 26-O-β-glucosidase (an enzyme involved in steroid saponin synthesis) was successfully identified by mass spectrometry analysis and de novo sequencing of a 2DE spot from an Agave attenuata sample.

Keywords

Agave CAM plants Protein extraction Protease Electrophoresis Mass spectrometry 

Abbreviations

2DE

Bidimensional electrophoresis

CAM

Crassulacean acid metabolism

TCA

Trichloroacetic acid

β-ME

β-mercaptoethanol

PMSF

Phenylmethylsulfonyl fluoride

Notes

Acknowledgements

We are thankful to Dr. Salvador Arias from Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México for providing O. ficus-indica, L.marginatus, and M. magnimamma samples. We also thank Unidad de Proteómica and Unidad Universitaria de Apoyo Bioinformático, Instituto de Biotecnología, Universidad Nacional Autónoma de México for all mass spectrometry analysis and the production of our local Agave fasta database, respectively. This work was supported by research grants from PAPIIT/DGAPA/UNAM IN212116 (F Lledías) and IG200515 (J Nieto-Sotelo and G Cassab), UNAM-Allied/Domecq P-150 (J Nieto-Sotelo and G Cassab), and CONACyT PN-247732 (J Nieto-Sotelo and G Cassab).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflicts of interest.

Human participants and animals

This article does not contain any studies with human participants or animals performed by any of the authors.

Supplementary material

10930_2017_9720_MOESM1_ESM.pdf (801 kb)
Supplementary material 1 (PDF 801 KB)

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Copyright information

© Springer Science+Business Media New York 2017

Authors and Affiliations

  1. 1.Departamento de Biología Molecular de Plantas, Instituto de BiotecnologíaUniversidad Nacional Autónoma de MéxicoCuernavacaMexico
  2. 2.Jardín Botánico, Instituto de BiologíaUniversidad Nacional Autónoma de MéxicoCiudad de MéxicoMexico

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