Submitted to: Proteomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: March 1, 2009
Publication Date: June 1, 2009
Citation: Krishnan, H.B., Oehrle, N.W., Natarajan, S.S. 2009. A Rapid and Simple Procedure for the Depletion of Abundant Storage Proteins from Legume Seeds to Advance Proteome Analysis: A Case Study Using Glycine Max. Proteomics. 9:3174-3188. Interpretive Summary: Proteomic analysis of plant tissue is inherently difficult, requiring great attention to tissue isolation, handling and manipulation; and more importantly, protein isolation, preparation, storage and separation techniques. Isolation of all the different types of plant tissues for a proteomic analysis using standard, or even logically adapted protocols, can unintentionally reduce the amount of protein harvest or unknowingly introduce protein modifications, ultimately making the final step, protein identification, difficult or impossible. Proteomic analysis of certain types of plant tissues, such as seed, leaf or tuber, is made even more difficult largely due to several or possibly many highly abundant proteins. This limits the yield of those inconspicuous nonabundant proteins. In this basic study we have developed a simple, fast, and inexpensive method to remove the majority of storage proteins from a seed extract than would significantly enhance the study of the nonabundant proteins within seeds. Our methodology would aid researchers in the isolation of more nonabundant seed proteins allowing for the discovery of new or novel proteins within the seed proteome, and allowing more clues about their nutritive value, yield potential, and environmental stress responses to be ascertained.
Technical Abstract: Electrophoretic analysis of plant proteomes containing thousands of proteins, has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the nonabundant proteins within seeds, using two-dimensional electrophoretic separation, is difficult when the overwhelming majority (60 – 80%) can be made up of storage proteins. However, dynamic resolution can be improved through fractionation of the proteome using separation techniques based upon different physiological or biochemical principles. We have thus developed a fast and simple fractionation technique using Ca2+ to precipitate the two major soybean (Glycine max) seed storage proteins, glycinin and ß-conglycinin (globulins). This method is highly effective, capable of removing 98% of the highly abundant soybean seed proteins from the seed extract, 91% of which are globulins. This ultimately allowed for 541 previously inconspicuous nonabundant proteins present in soybean seed to be more detectable (volume increase of less than 50%) using fluorescent detection and therefore more available for in-gel analysis. Of those 541 enhanced spots, 250 increased more than 3-fold when detected using SYPRO stain, a stain conducive to obtaining spots for isolation and identification. Ca2+ fractionation also made evident 63 phosphorylated proteins previously undetected, and enhanced the visibility of 15 phosphorylated proteins, using two-dimensional electrophoretic separation and phosphoprotein in-gel stain analysis. Application of this methodology towards other legumes such as peanut, bean, pea, vetch, alfalfa and others, also containing high amounts of globulin storage proteins, was examined and is reported here.