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Assessing factors for reliable quantitative proteomics based on two‐dimensional gel electrophoresis

2004; Wiley; Volume: 4; Issue: 7 Linguagem: Inglês

10.1002/pmic.200300731

1615-9861

Julie Fiévet, Christine Dillmann, Gilles Lagniel, Marlène Davanture, Luc Négroni, Jean Labarre, Dominique de Vienne,

Mass Spectrometry Techniques and Applications

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.