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

Abstract

2-D analysis of plant proteomes containing thousands of proteins has limited dynamic resolution because only abundant proteins can be detected. Proteomic assessment of the non-abundant proteins within seeds is difficult when 60-80% is storage proteins. Resolution can be improved through sample fractionation using separation techniques based upon different physiological or biochemical principles. We have developed a fast and simple fractionation technique using 10 mM Ca(2+) to precipitate soybean (Glycine max) seed storage globulins, glycinin and beta-conglycinin. This method removes 87+/-4% of the highly abundant seed proteins from the extract, allowing for 541 previously inconspicuous proteins present in soybean seed to be more detectable (volume increase of >or=50%) using fluorescent detection. Of those 541 enhanced spots, 197 increased more than 2.5-fold when visualized with Coomassie. The majority of those spots were isolated and identified using peptide mass fingerprinting. Fractionation also provided detection of 63 new phosphorylated protein spots and enhanced the visibility of 15 phosphorylated protein spots, using 2-D electrophoretic separation and an in-gel phosphoprotein stain. Application of this methodology toward other legumes, such as peanut, bean, pea, alfalfa and others, also containing high amounts of storage proteins, was examined, and is reported here.

Effects of proteome rebalancing and sulfur nutrition on the accumulation of methionine rich δ-zein in transgenic soybeans

“…Two-dimensional gel electrophoresis of soybean seed proteins were performed as described earlier (Krishnan et al, 2009). Coomassie stained gels were destained with multiple changes of ultrapure water to remove background and scanned using an Epson Perfection V700 scanner controlled through Adobe Photoshop.…”

Post-Genomics Studies of Developmental Processes in Legume Seeds

“…For the entire seed, the citations refer to post-genomics experiments carried out in various legume species at the level of the transcriptome (Firnhaber et al, 2005;Buitink et al, 2006;Gallardo et al, 2007;Benedito et al, 2008;Verdier et al, 2008), metabolome (Vigeolas et al, 2008), ionome (Sankaran et al, 2009), and proteome. At the level of the proteome, some citations refer to the mature seed (Watson et al, 2003;Magni et al, 2007;Bourgeois et al, 2009;Krishnan et al, 2009;Natarajan et al, 2009), to the kinetics of seed development (Hajduch et al, 2005;Gallardo et al, 2007;Agrawal et al, 2008;Dam et al, 2009), and to a functional study for identifying targets of thioredoxin (Alkhalfioui et al, 2007). For the separated seed tissues, the citations refer to transcriptome and proteome analyses in soybean (Le et al, 2007) and M. truncatula (Boudet et al, 2006;Zhang et al, 2006;Gallardo et al, 2007).…”

Using proteomics to study sexual reproduction in angiosperms

“…Multiple strategies for removal of abundant proteins are available, including 0 10 20 30 Literature reports Fig. 4 Numerical analysis of literature reports describing use of a proteomics-based strategy, as a function of reproductive tissue/organ/ stage differential solubility, affinity chromatography, and immuno-removal (Miernyk and Johnston, 2006;Li et al 2008;Krishnan et al 2009). Application of these methods, further refinements, and ingenious experimental designs will allow a much greater depth of analysis to studies of plant reproduction and help us to better understand this fascinating but extremely complex process.…”