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

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An Erratum to this article was published on 19 October 2017

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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.

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Change history

  • 19 October 2017

    The original version of this article unfortunately contains a mistake. The authors have inadvertently incorrectly listed the concentration of TCA in the acetone/TCA/β-ME solution in the materials and methods section of this paper. The TCA concentration in Sects. 2.3.2 and 2.3.5 should be 10% TCA, making the solution acetone/10% TCA/0.07% β-ME. It is now corrected with this erratum.

Abbreviations

2DE:

Bidimensional electrophoresis

CAM:

Crassulacean acid metabolism

TCA:

Trichloroacetic acid

β-ME:

β-mercaptoethanol

PMSF:

Phenylmethylsulfonyl fluoride

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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).

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Authors and Affiliations

  1. Departamento de Biología Molecular de Plantas, Instituto de Biotecnología, Universidad Nacional Autónoma de México, Av. Universidad # 2001, Col. Chamilpa C.P., 62210, Cuernavaca, Morelos, Mexico

    Fernando Lledías, Viridiana Rivas & Gladys I. Cassab

  2. Jardín Botánico, Instituto de Biología, Universidad Nacional Autónoma de México, Tercer Circuito Exterior, s/n, Coyoacán, Cd. Universitaria, 04510, Ciudad de México, Mexico

    Felipe Hernández, Abisaí García-Mendoza & Jorge Nieto-Sotelo

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  1. Fernando Lledías
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  2. Felipe Hernández
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  3. Viridiana Rivas
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  4. Abisaí García-Mendoza
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  5. Gladys I. Cassab
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  6. Jorge Nieto-Sotelo
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Correspondence to Jorge Nieto-Sotelo.

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An erratum to this article is available at https://doi.org/10.1007/s10930-017-9739-5.

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Lledías, F., Hernández, F., Rivas, V. et al. A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis. Protein J 36, 308–321 (2017). https://doi.org/10.1007/s10930-017-9720-3

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