Biology Reference
In-Depth Information
Chapter 11
2DE Analysis of Forest Tree Proteins Using Fluorescent
Labels and Multiplexing
Jenny Renaut , Céline Leclercq , and Sébastien Planchon
Abstract
Although proteomists working with gel-free methods are considering the gels as coming from the past,
proteomics based on gels has still a lot of opportunities to offer and acquisition of images on which thou-
sands of spots may be resolved is still largely performed. Nowadays, two-dimensional electrophoresis
remains a powerful tool to explore the plant proteome and to unravel changes in protein abundance
between samples. Some weak points can be pointed out, as for any method, as for example the lack of
reproducibility, or the detection of low-abundance proteins. The use of the technique called “difference
gel electrophoresis” or “DIGE” can help to overcome or at least to reduce these inconveniences. DIGE
requires the labelling of proteins by fl uorochromes prior to their separation on 2DE gels. This technique
may be applied to a wide array of plant stress studies, among others to trees. Accurate quantitative results
can then be obtained and proteins presenting an interest in the studied stress are subsequently subjected
to an enzymatic digestion (usually with trypsin) and identifi ed using electrospray ionization, matrix-
assisted laser desorption/ionization-time-of-fl ight-MS, and/or tandem MS.
Key words
Two-dimensional electrophoresis (2DE), Quantitative approach, DIGE, Protein abun-
dance, Trees
1
Introduction
Proteomics, i.e., the study of the protein complement of the genome
[
1
], is one of the high-throughput techniques of the post-genome
era, such as transcriptomics, metabolomics, and phenomics, taking
advantage of the completion of several sequenced genomes (e.g.,
Arabidopsis, rice, poplar) [
2
-
4
]. Proteomics allows to study plants
at different stages of their life cycle, and to directly relate the pro-
teins and their functions in plants. Two main approaches are used:
the gel-based approach (e.g., 2DE) and the gel-free analyses (e.g.,
LC-MS/MS), each presenting advantages and inconveniences. In
plants and especially for trees, a majority of the results are still
provided by the use of classical 2DE (
see
[
5
,
6
] for a review).
