Submitted to: Journal of Basic and Applied Sciences
Publication Type: Peer reviewed journal
Publication Acceptance Date: 4/23/2013
Publication Date: 4/25/2013
Publication URL: http://handle.nal.usda.gov/10113/57024
Citation: Natarajan, S.S., Krishnan, H.B., Khan, F.H., Chen, X., Garrett, W.M., Lakshman, D.K. 2013. Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry. Journal of Basic and Applied Sciences. 9:309-332. Interpretive Summary: Soybean provides an inexpensive source of protein for both humans and animals. Genetic modification (GM) serves as a potential tool to improve the quality and quantity of soybean seed components. Soybean embryonic tissue provides a suitable experimental material for examining the expression of foreign genes (transgenes) that are used to create GM soybeans. However, analytical methods to accurately determine the identity, quantity and composition of proteins in GM soybean remain a challenge. We have standardized and applied available technologies to determine and quantify the range of proteins present in the embryo of normal, nontransgenic soybeans in order to create a “protein reference map”. To do this we extracted embryo proteins, then separated and identified the proteins using a technique called “peptide mass fingerprinting”. Of a total of 401 proteins separated, 335 proteins were identified. This approach will be important in providing a way to determine protein composition and variation in GM soybeans. The protein reference map that we developed provides scientists with a basis for comparisons with similar maps derived from GM other soybean tissues.
Technical Abstract: A proteomic approach based on two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) for protein separation and subsequent mass spectrometry (MS) for protein identification was applied to establish a proteomic reference map for the soybean embryonic axis. Proteins were extracted from dissected embryonic axes and separated in the first dimension using a pH range from 4-7. A total of 401 protein spots were isolated, digested with trypsin, and analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). We identified 335 protein spots by searching NCBI non redundant databases using the Mascot search engine and found a total of 200 unique proteins. Gene Ontology (GO) analysis was employed to understand the molecular processes in which the identified embryonic axes proteins are involved. The majority of proteins play a functional role in catalytic activity (42.9%) and binding (39.3%), followed by nutrient reservoir activity (5.3%), structural molecular activity (4.0%), antioxidant activity (3.2%), transporter activity (2.4%), enzyme regulator activity (1.2%), molecular transducer activity (0.8%), and transcription regulator activity (0.8%). The results indicate that 2D-PAGE, combined with LC-MS/MS, is a sensitive and useful technique for separation and identification of soybean low and high abundant embryonic axis proteins.