TY - JOUR
T1 - Comparison of two label-free global quantitation methods, APEX and 2D gel electrophoresis, applied to the Shigella dysenteriae proteome
AU - Kuntumalla, Srilatha
AU - Braisted, John C.
AU - Huang, Shih Ting
AU - Parmar, Prashanth P.
AU - Clark, David J.
AU - Alami, Hamid
AU - Zhang, Quanshun
AU - Donohue-Rolfe, Arthur
AU - Tzipori, Saul
AU - Fleischmann, Robert D.
AU - Peterson, Scott N.
AU - Pieper, Rembert
N1 - Funding Information:
This work was funded by the Pathogen Functional Genomics Resource Center (PFGRC), through a contract awarded by the National Institutes of Allergy and Infectious Diseases (NIAID), contract No. N01-AI-15447, awarded to the J. Craig Venter Institute (JCVI), Rockville, Maryland, USA. We wish to thank Christine Vogel and Edward M. Marcotte from the University of Texas at Austin for extremely helpful and extensive discussions regarding the APEX methodology. At Tufts University, this project was funded in whole or in part with Federal funds from the NIAID, NIH, DHHS, under contract number N01-AI-30050.
PY - 2009/6/29
Y1 - 2009/6/29
N2 - 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 genome-predicted 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.
AB - 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 genome-predicted 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.
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U2 - 10.1186/1477-5956-7-22
DO - 10.1186/1477-5956-7-22
M3 - Article
C2 - 19563668
AN - SCOPUS:68349107865
SN - 1477-5956
VL - 7
JO - Proteome Science
JF - Proteome Science
M1 - 22
ER -