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ARS Home » Northeast Area » Beltsville, Maryland (BARC) » Beltsville Agricultural Research Center » Soybean Genomics & Improvement Laboratory » Research » Publications at this Location » Publication #273924

Title: Analysis of soybean embryonic axis proteins by two-dimensional gel electrophoresis and mass spectrometry

item Natarajan, Savithiry - Savi
item Krishnan, Hari
item Khan, Farooq
item Oehrle, Nathan
item CHEN, XI - Nanjing Agricultural University
item Garrett, Wesley

Submitted to: Journal of Basic and Applied Sciences
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/22/2012
Publication Date: 1/10/2013
Citation: Natarajan, S.S., Krishnan, H.B., Khan, F.H., Oehrle, N.W., Chen, X., Garrett, W.M. 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) of soybean has become inevitable because of the need for more nutritious and high quality soybean products. 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 embryonic axis of normal, nontransgenic soybeans in order to create a “protein reference map” of the soybean embryonic axis. To do this we extracted embryonic axis proteins, then separated and identified the proteins using a technique called “peptide mass fingerprinting”. Of a total of 401 proteins separated, 336 proteins were identified using mass spectrometry and database searches. 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 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 axis and separated 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 have identified 336 protein spots by searching NCBI non redundant databases using the Mascot search engine. Out of 336 proteins, we have identified a total of 201 unique proteins. We employed Gene Ontology (GO) analysis to understand the molecular processes in which the identified embryonic axis proteins are involved. The majority of the proteins are involved in catalytic activity (43.1%) and binding (39.1%) followed by nutrient reservoir activity (5.2%), structural molecule 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 embryonic axis proteins.