Submitted to: Phytochemistry
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/18/2006
Publication Date: 6/18/2006
Citation: Xu, C., Garrett, W.M., Sullivas, J., Caperna, T.J., Natarajan, S.S. 2006. Separation and identification of soybean leaf proteins by two-dimensional gel electrophoresis and mass spectrometry. Phytochemistry. 67: 2431-2440. Interpretive Summary: Soybean provides an inexpensive source of protein for both humans and animals. Genetic modification (GM) of soybean has become inevitable because of the need for more nutritious and high quality soybean products. Leaf tissue provides suitable experimental material for examining the expression of transgenes (genes from other plants or organisms) that are used to create GM soybean. However, analytical methods to accurately determine the identity, quantity and composition of proteins in GM soybean remain a challenge. Therefore, we have standardized and applied available technologies to determine and quantify the spectrum of proteins present in the leaves of normal soybean that contain no transgenes. To do this we have extracted leaf proteins, then separated and identified the proteins using a technique called “peptide mass fingerprinting”. Of a total of 260 proteins separated, 119 proteins were identified using three protein databases. This approach will be important in providing a way to determine protein composition and variation in GM soybeans and to create a “protein reference map” of the soybean leaf. The protein reference map that we developed provides scientists with a basis for comparisons with similar maps derived from GM soybean.
Technical Abstract: To establish a proteomic reference map for soybean leaves, we separated and identified leaf proteins using two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS). Tryptic digests of 260 spots were subjected to peptide mass fingerprinting (PMF) by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) MS. Fifty-three protein spots were identified by searching NCBInr and SwissProt databases using the Mascot search engine. Sixty-seven spots that were not identified by MALDI-TOF-MS analysis were analyzed with liquid chromatography tandem mass spectrometry (LC-MS), and sixty-six of these spots were identified by searching against the NCBInr, SwissProt and expressed sequence tag (EST) databases. The majority of the identified leaf proteins are involved in energy metabolism. The results indicate that 2D-PAGE, combined with MALDI-TOF- MS and LC-MS, is a sensitive and powerful technique for separation and identification of soybean leaf proteins. A summary of the identified proteins and their putative functions is discussed.