Proteomics studies are most often carried out using 2D-PAGE and protein staining. However, protein staining only gives information on the steady-state accumulation levels of proteins. For a time-dependent process, it might be advantageous to know which are the proteins that are synthesized during the process and to which extent they accumulate. In this case characterizing the de novo synthesized proteome is the method of choice. We have devised a protocol to characterize the de novo synthesized proteome from germinating Arabidopsis seeds (Rajjou et al., 2004, 2006).
For this purpose, labeled proteins are synthesized in vivo by seeds imbibed on water (see Germination assays) in the presence of [35S]-Met (1.85 MBq; ICN Biomedicals SARL). Following incubation, protein extracts are prepared (see Preparation of total protein extracts) and protein synthesis is measured by TCA precipitation of aliquots of reaction mixtures spotted on Whatmann GF/C filters; after eight washing steps in cold 5% TCA and 0.04 M sodium pyrophosphate and two washing steps in absolute methanol, filters are dried and counted for radioactivity in a liquid scintillation counter (Dietrich et al., 1985). It is recommended to carry out this simple assay prior to the 2D-PAGE analyses, since it allows checking the extent of labeled Met precursor incorporation into the proteins synthesized de novo. Protein extracts can then be submitted to 2D gel electrophoresis as described in 2D electrophoresis. Proteins on the 2D gels are stained by silver nitrate (Protein staining and analysis of 2D gels). Then, stained 2D gels are dried for 2 days at room temperature in a sandwich composed of, from bottom to top: one sheet of cellophane model Gel Dryer (Bio-Rad), 2D gel, one sheet of Saran wrap (VWR international SAS), and one sheet of cellophane model Gel Dyer (Bio-Rad). After drying, the upper sheet of cellophane and the Saran wrap sheet are peeled and gels are submitted to Phosphorimager analysis (Molecular Dynamics Storm 840 phosphorimager, Amersham Biosciences). Labeled 2D protein patterns are scanned as described above for the silver-nitrate-stained gels. Relative protein neosynthesis levels are quantitated by densitometric analyses of the spots on the autoradiography as described in Protein staining and analysis of 2D gels.
Comparison of 2DE profiles of total proteins revealed by silver nitrate (A) and proteins synthesized de novo and revealed by autoradiography (B). Proteins are extracted from germinating Arabidopsis seeds (24-h imbibition) on water and [35S]-Met. (A) 2DE gels stained with silver nitate. (B) Autoradiography of proteins radiolabeled in the presence of [35S]-Met. (C) Composite gel with protein spots shown in false colors. Red, proteins revealed by autoradiography but not by silver nitrate. Green, proteins revealed by silver staining but not by autoradiography. Blue, proteins revealed both by silver staining and autoradiography. Data from Rajjou L (2006, PhD thesis).
Rajjou L, Huguet R, Robin C, Belghazi M, Job C, Job D (2006) Proteomic investigation of the effect of salicylic acid on Arabidopsis seed germination and establishment of early defense mechanisms. Plant Physiology 141, 910-923.
Rajjou L, Gallardo K, Debeaujon I, Vandekerckhove J, Job C, Job D (2004) The effect of α-amanitin on the Arabidopsis seed proteome highlights the distinct roles of stored and neosynthesized mRNAs during germination. Plant Physiology 134, 1598-1613.