Comparison of two label-free global quantitation methods , APEX and 2 D gel electrophoresis , applied to the Shigella dysenteriae proteome

The in vitro stationary phase proteome of the human pathogen Shigella dysenteriae serotype 1 (SD1) was quantitatively analyzed in Coomassie Blue G250 (CBB)-stained 2D gels. More than four hundred and fifty proteins, of which 271 were associated with distinct gel spots, were identified. In parallel, we employed 2D-LC-MS/MS followed by the label-free computationally modified spectral counting method APEX for absolute protein expression measurements. Of the 4502 genomepredicted SD1 proteins, 1148 proteins were identified with a false positive discovery rate of 5% and quantitated using 2D-LC-MS/MS and APEX. The dynamic range of the APEX method was approximately one order of magnitude higher than that of CBB-stained spot intensity quantitation. A squared Pearson correlation analysis revealed a reasonably good correlation (R2 = 0.67) for protein quantities surveyed by both methods. The correlation was decreased for protein subsets with specific physicochemical properties, such as low Mr values and high hydropathy scores. Stoichiometric ratios of subunits of protein complexes characterized in E. coli were compared with APEX quantitative ratios of orthologous SD1 protein complexes. A high correlation was observed for subunits of soluble cellular protein complexes in several cases, demonstrating versatile applications of the APEX method in quantitative proteomics. Introduction Until recently, quantitative proteomics studies have mainly relied on two-dimensional (2D) gel electrophoresis combined with protein identification by mass spectrometry (MS) to analyze large datasets of proteins from complex protein mixtures [1,2]. Quantitation of relative protein abundances from 2D gels has involved the comparison of protein spot intensities across two or more Published: 29 June 2009 Proteome Science 2009, 7:22 doi:10.1186/1477-5956-7-22 Received: 10 April 2009 Accepted: 29 June 2009 This article is available from: http://www.proteomesci.com/content/7/1/22 © 2009 Kuntumalla et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.