Assessing factors for reliable quantitative proteomics based on two-dimensional gel electrophoresis.
We statistically analysed various factors to get accurate estimates of protein quantities from two-dimensional gels. Yeast proteins were labelled with (35)S or stained with Coomassie Brilliant Blue G-250, and spots were automatically quantified with software packages Kepler, ImageQuaNT, Melanie 3.0 and Progenesis. The different software packages proved to have very similar performances. With (35)S-labelled actin spot as a reference, we studied the staining efficiency of colloidal Coomassie blue as a function of amino acid composition of the protein, and derived an equation to estimate the number of molecules per cell from blue-stained proteins. Absolute quantification of most glycolytic enzymes was carried out in two yeast strains.
Fiévet
Julie
J
UMR de Génétique Végétale, INRA/UPS/CNRS/INA-PG, Ferme de Moulon, F-91190 Gif-sur-Yvette, France.
Dillmann
Christine
C
Lagniel
Gilles
G
Davanture
Marlène
M
Negroni
Luc
L
Labarre
Jean
J
de Vienne
Dominique
D
D000199:Actins / Q000737:chemistry; D002853:Chromatography, Liquid; D004396:Coloring Agents / Q000494:pharmacology; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D005656:Fungal Proteins / Q000737:chemistry*; D006019:Glycolysis; D007091:Image Processing, Computer-Assisted; D007700:Kinetics; D013058:Mass Spectrometry; D015233:Models, Statistical; D020543:Proteome / Q000737:chemistry; D040901:Proteomics / Q000379:methods*; D012394:Rosaniline Dyes / Q000494:pharmacology; D012984:Software; D013462:Sulfur Radioisotopes; D015003:Yeasts / Q000378:metabolism
D000199:Actins; D004396:Coloring Agents; D005656:Fungal Proteins; D020543:Proteome; D012394:Rosaniline Dyes; D013462:Sulfur Radioisotopes; C048139:Coomassie blue