Submitted to: Journal of Proteome Research
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/14/2009
Publication Date: 9/18/2009
Citation: Krishnan, H.B., Natarajan, S.S. 2009. A Simple Method for Rapid Depletion of Rubisco from Soybean (Glycine max) Leaf for Proteomic Analysis of Lower Abundance Proteins. Journal of Proteome Research. 70(17-18):1958-1964. Interpretive Summary: Proteomic analysis (analysis of the total protein complement of a tissue) of plant tissues is inherently difficult, requiring great attention to tissue isolation, handling and manipulation; and more importantly, protein isolation, preparation, storage and separation techniques. Isolation of proteins from all the different types of plant tissues for a proteomic analysis using standard, or even logically adapted protocols, can unintentionally reduce the amount of proteins harvested 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 because of the presence of several or many highly abundant proteins, which limit the yield of those less conspicuous but important nonabundant proteins. In this study we have developed a simple, fast and inexpensive method to remove the majority of the highly abundant CO2 fixation enzyme Rubisco from soybean leaf. Our simple extraction procedure will significantly enhance the study of the nonabundant proteins within leaf. Our methodology will allow for the discovery of new or novel proteins within the leaf proteome and will generate more clues as to their function, regulation and biotic and abiotic stress responses.
Technical Abstract: 2-DE analysis of complex plant proteomes has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the low abundance proteins within leaf tissue is difficult when it is comprised of 30 – 50% of the CO2 fixation enzyme Rubisco. Resolution can be improved through depletion of Rubisco using fractionation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca2+ and 10 mM phytate to precipitate Rubisco from soybean leaf soluble protein extract. This method is not only rapid, but also inexpensive, and capable of removing 85% of the extremely abundant Rubisco enzyme from soybean leaf soluble protein extract. This method allowed for roughly 230 previously inconspicuous protein spots in soybean leaf to be more easily detectable (3 fold increase in % volume) using fluorescent detection and allowed 28 phosphorylated proteins previously undetected, to be isolated and identified by MALDI-TOF MS.