| Description |
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Abstract: 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 ... read moreby 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 (R 2 = 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.
Keywords: two-dimensional electrophoresis, Absolute protein expression,
Attribute relation file format, Coomassie Brilliant Blue, False positive rate,
Isoelectric focusing, Laboratory Information Management System, Liquid chromatography
with tandem mass spectrometry, Matrix-assisted laser desorption ionization with
tandem time of flight, Mass spectrometry, Methyl methanethiosulfonate, protein
molecular weight, estimation of expected number of unique proteotypic peptides for a
given protein i, protein isoelectric point, squared Pearson correlation coefficient,
Spearman rank correlation coefficient, Room temperature, Shigella dysenteriae type 1,
Triethyl ammonium bicarbonate, (Tris(2-carboxyethyl)phosphine).
Springer Open.read less
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| Citation |
- Kuntumalla, Srilatha, John C. Braisted, Shih-Ting Huang,
Prashanth P. Parmar, David J. Clark, Hamid Alami, Quanshun Zhang, Arthur
Donohue-Rolfe, Saul Tzipori, Robert D. Fleischmann, Scott N. Peterson, and Rembert
Pieper. "Comparison of two label-free global quantitation methods, APEX and 2D gel
electrophoresis, applied to the Shigella dysenteriae proteome." Proteome Science 7,
no. 1 (12, 2009): 1-16.
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