A Rapid and Reliable Method for Total Protein Extraction from Succulent Plants for Proteomic Analysis
Por:
Lledias, Fernando, Hernandez, Felipe, Rivas, Viridiana, Garcia-Mendoza, Abisai, Cassab, Gladys I., Nieto-Sotelo, Jorge
Publicada:
1 ago 2017
Resumen:
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-beta-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.
Filiaciones:
Lledias, Fernando:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Biol Mol Plantas, Av Univ 2001, Cuernavaca 62210, Morelos, Mexico
Hernandez, Felipe:
Univ Nacl Autonoma Mexico, Jardin Bot, Inst Biol, Tercer Circuito Exterior S-N, Ciudad De Mexico 04510, Mexico
Rivas, Viridiana:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Biol Mol Plantas, Av Univ 2001, Cuernavaca 62210, Morelos, Mexico
Garcia-Mendoza, Abisai:
Univ Nacl Autonoma Mexico, Jardin Bot, Inst Biol, Tercer Circuito Exterior S-N, Ciudad De Mexico 04510, Mexico
Cassab, Gladys I.:
Univ Nacl Autonoma Mexico, Inst Biotecnol, Dept Biol Mol Plantas, Av Univ 2001, Cuernavaca 62210, Morelos, Mexico
Nieto-Sotelo, Jorge:
Univ Nacl Autonoma Mexico, Jardin Bot, Inst Biol, Tercer Circuito Exterior S-N, Ciudad De Mexico 04510, Mexico
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