A rapid method for depletion of Rubisco from soybean (Glycine max) leaf for proteomic analysis of lower abundance proteins.
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 CO(2) 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 Ca(2+) 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 vol%) using fluorescent detection and allowed 28 phosphorylated proteins previously undetected, to be isolated and identified by MALDI-TOF-MS.
Krishnan
Hari B
HB
Plant Genetics Research Unit, Agricultural Research Service, United States Department of Agriculture, 108 Curtis Hall, University of Missouri, Columbia, MO 65211, USA. Hari.Krishnan@ARS.USDA.GOV
Natarajan
Savithiry S
SS
D010936:Plant Extracts / Q000302:isolation & purification; D018515:Plant Leaves / Q000737:chemistry; D020543:Proteome; D040901:Proteomics / Q000379:methods*; D012273:Ribulose-Bisphosphate Carboxylase / Q000737:chemistry / Q000302:isolation & purification*; D030262:Soybean Proteins / Q000302:isolation & purification*; D013025:Glycine max / Q000737:chemistry*
D010936:Plant Extracts; D020543:Proteome; D030262:Soybean Proteins; D012273:Ribulose-Bisphosphate Carboxylase