A fast method for quantitative proteomics based on a combination between two-dimensional electrophoresis and 15N-metabolic labelling.
We provide a method for accurate protein quantitation that uses two-dimensional (2-D) gel electrophoresis for protein separation, but does not require extensive statistical analysis of staining intensities on gels. Instead, accurate quantitation occurs on the mass spectrometer (MAS) on multiple peptides to provide statistical evidence. In an example study, Sulfolobus solfataricus cells were grown on the carbon sources glucose, fructose and glutamate. The glucose phenotype (reference) was grown on (15)N-enriched medium. Next, the glutamate and the fructose phenotypes are mixed with the reference and two 2-D gels are created. Staining intensities of gel spots in this case are used for initial, semiquantitative assessment of differential expression. On this basis, spots are selected for accurate quantitation on the MAS. A number of differentially expressed proteins were found, for example: a (25.2 +/- 8.2)-fold upregulation of isocitrate lyase and a (7.14 +/- 0.82)-fold downregulation of glucose dehydrogenase on glutamate compared to glucose. With this protocol, intergel and interlaboratory comparisons are facilitated, since the light and heavy versions of a protein are equally affected by variations in sample preparation and buffer composition. Because the statistical evidence is gathered on the MAS, the need to run vast numbers of gels is removed.
Snijders
Ambrosius P L
AP
Biological and Environmental Systems Group, Department of Chemical and Process Engineering, University of Sheffield, Mappin Street, Sheffield S1 3JD, UK.
de Vos
Marjon G J
MG
de Koning
Bart
B
Wright
Phillip C
PC
D015536:Down-Regulation; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005632:Fructose / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D042843:Glucose 1-Dehydrogenase / Q000096:biosynthesis; D018698:Glutamic Acid / Q000378:metabolism; D007522:Isocitrate Lyase / Q000096:biosynthesis; D007553:Isotope Labeling; D013058:Mass Spectrometry / Q000379:methods*; D009587:Nitrogen Isotopes; D040901:Proteomics / Q000379:methods*; D048229:Sulfolobus solfataricus / Q000378:metabolism; D015854:Up-Regulation
D009587:Nitrogen Isotopes; D005632:Fructose; D018698:Glutamic Acid; D042843:Glucose 1-Dehydrogenase; D007522:Isocitrate Lyase; D005947:Glucose