conference schedule and program with abstracts - Horticulture ...

P-31
Grape berry development proteome: protein extraction and resolution by two-dimensional
gel electrophoresis
R. Paul 1 , M. Jego 1 , C. Muñoz 1 , A.M. Almeida 1 , P. Hinrichsen* 2 , A. Orellana 1
1 Centro de Biotecnología Vegetal, Universidad Andrés Bello, La Serena, Chile; 2 INIA La
Platina, Santiago, Chile
*Corresponding author: phinrichsen@inia.cl
Proteomic analyses have emerged in several areas of plant biology. Two-dimensional gel
electrophoresis (2-DE) coupled with mass spectrometry is one of the most efficient and powerful
methods to study complex patterns of gene expression at the protein level. Nevertheless, the
major constraint of 2-D protein separation is the quality of the protein extracts to be assayed. The
electrophoretic separation of proteins from plant tissue extracts is often complicated by other
non-protein contaminants such as organic acids, lipids, polyphenols, pigments, terpenes, and
other secondary metabolites. High quality whole cell protein extracts from woody plants,
especially grapevine, is difficult to obtain due to the high levels of contaminants. The objective
of this work was to develop protein extraction protocols to ensure a high-resolution twodimensional
gel electrophoresis for proteomic profiling from berries at different development
stages: fruit set, 2-4 mm and 6-8 mm berries, pre- and post-véraison stages. It uses a mixed
protocol of classical TCA/acetone precipitation and phenol extraction. The pre-treatment with
TCA/acetone is a very effective precipitant, eliminating instantly proteolytic and other modifying
enzymes. On the other hand phenol extraction optimizes extraction of cytosolic and membrane
proteins. The high pH buffer (7.8) inhibits most common proteases, assures that the abundant
phenolic compounds are mainly ionized, and neutralizes acids that are released by disrupted
vacuoles. The efficiency of this combined protocol was monitored by two-dimensional gel
electrophoresis. Protein was loaded on isoelectric strip gels in which the pH gradient was
established with pH 4-7 ampholines (Multiphor II system). The second dimension was carried
out on 12% SDS-polyacrylamide gels, and proteins were visualized by deep purple staining. This
protocol is being used to extract protein for proteomic analysis from a ‘Ruby seedless’ x
‘Thompson seedless’ crossing which exhibited extreme and opposite phenotypes for seed and
berry size in the scope of a Chilean private-public partnership, the Biofrutales Consortium.
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