Proteomics is the study of the structure, function and interaction of proteins. Proteins play a critical role in the life of any organism by being involved in many processes within the cell. The proteome, which is the complete collection of proteins in an organism, is much more complicated to study than the genome or transcriptome. An organism harboring 20 000 genes can have more than 1 million different proteins due to alternative splicing and post-translational modifications. Furthermore, the level of transcription of a gene often does not reflect the level of the encoded protein. An mRNA can be present in numerous copies in a cell but the mRNA stability (half life) and efficiency of translation will affect the level of the corresponding protein. A protein can also be present in a cell but inactive until subjected to post-translational modifications like phosphorylation. Finally, many proteins can interact with other proteins and only the resulting complex may have biological function. The measurement of mRNA levels via transcriptomics (e.g., via the use DNA microarrays, see discussion of this subject on our website) is complimentary to proteomics.
Figure 1. Picture of soybean root hairs. An infection thread is visible due to blue staining of the infecting bacterium, B. japonicum.
Figure 2. 2 D-PAGE electrophoresis on proteins from root hair
Root hairs are single, tubular-shaped cells (Figure 1) formed from the differentiation of epidermal cells, called trichoblasts, on primary and secondary roots. Root hairs improve the capacity of the root to absorb water and nutrients from the soil by increasing the surface area of the root. Root hairs are also the site of the infection of legumes by rhizobia leading to the establishment of a nitrogen fixing symbiosis (i.e., nodulation). As part of our effort to explore the early steps in symbiotic infection, we are establishing a root hair proteome reference map for soybean (Glycine max) by using 2D-SDS PAGE gel electrophoresis (Figure 2) and MudPIT (Multidimensional Protein Identification Technology). Proteins extracted from soybean root hairs were separated by 2D gel electrophoresis. One thousand and two spots were picked. Tryptic peptides were analyzed on an Applied Biosystems 4700 MALDI TOF/TOF. Proteins identified by both approaches are mainly involved in primary metabolism, protein synthesis and processing, defense response and stress, and signal transduction.
Figure 3. A schematic of the procedures followed to identify proteins differentially expressed in root hairs after inoculation with B. japonicum. Proteins identified in this way are then identified following mass spectrometry.
SpotLink: Tool for proteomics data organization and visualization.
As descrobed in our publication (Brechenmacher et al., 2009), proteomic analysis was performed on soybean root hairs inoculated or unnoculated with Bradyrhizobium japonicum at different time points including 0, 12, 24, 36, 48, 72 and 96 hours post-inoculation in order to identify proteins having a critical role in the early stage of nodulation. The root hair proteome of the symbiotic interaction was analyzed by 2-D PAGE, followed by MS/MS, as well as using the MudPIT approach. The spotlink tool above gives you access to the results of the 2-D gel analysis. A full list of all identified proteins is available in the Brechenmacher et al., (2009) publication as supplemental table S1. This work builds upon our earlier study (Wan et al., 2005).
Current efforts are focused on a large scale proteomic analysis utilizing a liquid chromatography-MS/MS approach. We are also currently analyzing phosphoproteome data obtained as part of this same study.
Check this website periodic posting of our proteomic data.
Wan JR, Torres M, Ganapathy A, Thelen J, DaGue BB, Mooney B, Xu D, Stacey G. Proteomic analysis of soybean root hairs after infection by Bradyrhizobium japonicum. Mol. Plant-Microbe Int. 2005, 18:458-467.
Brechenmacher L, Lee J, Sachdev S, Song Z, Nguyen THN, Trupti J, Dague B, Oehrle N, Libault M, Mooney B, Xu D, Cooper B, Stacey G Establishment of a protein reference map for soybean root hair cells.Plant Physiol.2009, 149: p. 670.