Although gene expression profiling can show all of the genes that respond during a cellular event, the procedure fails to reveal exactly what directed the expression changes in the first place. In reality, these important initial responses to an external stimulus usually are not mediated by gene expression changes. Rather they are precipitated by a number of already present proteins that are activated by the stimulus, and consequently activate other proteins in a signal cascade or they stimulate gene expression directly. For example, reversible phosphorylation is one mode of controlling proteins involved in plant development and stress response.
Mass spectrometry (MS) is a useful method for identifying proteins. Coupled with high-throughput screening techniques, thousands of proteins can be identified. Protein accumulation levels between samples can also be measured. The NCSB scientists are using MALDI-TOF-TOF MS and LC-MS techniques to identify proteins from soybean leaves, roots, root hairs, and seeds collected at different developmental stages as well as from plants subjected to biotic stress (e.g. soybean cyst nematode, rust disease) and abiotic stresses (e.g. drought and temperature). Also, proteins were resolved on 2-dimensional gels and in some cases. Fluorescent labeling of the proteins and the comparison of proteins from treated and control samples will help resolve and quantify the differential accumulation of proteins associated with each tissue, developmental stage or stress response. The next step is the excision of the separated proteins the gel and analyze by MS. Major goal of these approach at the NCSB is to build a general catalogue of proteins that accumulate in these samples and to build tissue specific and stress specific proteome map of soybean. Further information about the NSF plant genome project on root hair proteome (http://www.soyroothair.org) and seed proteome (http://www.oilseedproteomics.missouri.edu/) can be obtained from the respective web sites.
Also, the NCSB scientists are working on the differential accumulation of phosphorylated proteins in developing or stressed tissues using quantitative phosphoproteomics techniques. The protein sequences will be deduced with the aid of soybean and other plant gene sequence data and available computer programs. Kinases, transcription factors and metabolic enzymes are some of the types of proteins that are expected to be found in these studies. Phosphorylated kinases and transcription factors will be considered to be candidates for controlling the plant cell during the developmental events or stress responses. Conducting protein profiling using various technologies help the soybean researchers to gain a comprehensive understanding of the changes in protein expression levels during plant development and stress conditions which will be compared to the transcript and phenome levels.
SPOTLINK : For Soybean
A tool for organizing and visualization of proteomics data.
Proteomics involves the analysis of complete set of proteins, such as identification or quantification of proteins, determination of their localization, interactions, function and etc.. During the course of experiment, data of various types are generated. These data need to be well organized for reference or further study. For this purpose, we developped SpotLink, which is a computer software for organizing proteomics data. It produces web pages that makes the data available to public. The output of SpotLink includes 1, a clickable 2D gel page that link to different identified protein spots. 2, two styles of web pages that contains the detailed information. Style A is for the protein expression data in terms of different time stamp. Styple B contains a summary page for spot and two pages for PMF data and MS/MS data respectively.