2DE analysis of forest tree proteins using fluorescent labels and multiplexing
J. Renaut, C. Leclercq, and S. Planchon
Methods in Molecular Biology, vol. 1072, pp. 141-154, 2014
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 thousands 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 fluorochromes 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 identified using electrospray ionization, matrix-assisted laser desorption/ionization-time-of-flight-MS, and/or tandem MS.