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

• Published in: Proteomics (Weinheim) Vol. 4; no. 7; pp. 1939 - 1949
• Main Authors: Fiévet, Julie, Dillmann, Christine, Lagniel, Gilles, Davanture, Marlène, Negroni, Luc, Labarre, Jean, de Vienne, Dominique
• Format: Journal Article
• Language: English
• Published: Weinheim WILEY-VCH Verlag 01.07.2004; WILEY‐VCH Verlag; Wiley-VCH; Wiley-VCH Verlag
• Subjects: Actins - chemistry; Analytical, structural and metabolic biochemistry; Biochemistry, Molecular Biology; Biological and medical sciences; Chromatography, Liquid; Colloidal Coomassie blue; Coloring Agents - pharmacology; Electrophoresis, Gel, Two-Dimensional - methods; Fundamental and applied biological sciences. Psychology; Fungal Proteins - chemistry; Glycolysis; Image analysis; Image Processing, Computer-Assisted; Kinetics; Life Sciences; Mass Spectrometry; Miscellaneous; Models, Statistical; Proteins; Proteome - chemistry; Proteomics - methods; Quantitative proteomics; Rosaniline Dyes - pharmacology; Software; Sulfur Radioisotopes; Two-dimensional gel electrophoresis; Yeast; Yeasts - metabolism; Proteomics; Two dimensional electrophoresis; Quantitative analysis
• ISSN: 1615-9853; 1615-9861
• DOI: 10.1002/pmic.200300731

We statistically analysed various factors to get accurate estimates of protein quantities from two‐dimensional gels. Yeast proteins were labelled with 35S 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 35S‐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.