Abundance of Malate dehydrogenase [Mdh] on acetate medium & Pyridoxal phosphate phosphatase [YbhA] 3 d into stationary phase

Range Mdh on acetate ~92,000: YbhA 3 days into stationary phase 2 proteins/cell
Organism Bacteria Escherichia coli
Reference Schmidt A et al., The quantitative and condition-dependent Escherichia coli proteome. Nat Biotechnol. 2015 Dec 7. doi: 10.1038/nbt.3418. p.2 left column 2nd paragraphPubMed ID26641532
Method P.2 caption to figure 1:"Workflow of system-wide protein abundance determination: The workflow comprised three steps. First, cells of the various samples were lysed and proteins extracted and proteolyzed using trypsin. The peptide mixtures were either further fractionated using OFFGEL electrophoresis (OGE) or directly analyzed in biological triplicates by shotgun LC-MS/MS and quantified by label-free quantification. Second, the cellular concentrations of 41 proteins covering all components of the glycolysis pathway were determined across all samples by SRM and SID [see comments section]. Therefore, for each protein, heavy labeled reference peptides (selected from the shotgun LC-MS/MS experiment) were synthetized. After spiking known amounts of these references into each sample, absolute quantities were determined for the corresponding proteins by SRM. Third, the numbers of cells taken for LC-MS/MS analyses were determined for each sample by flow cytometry. With this information, the protein concentrations determined by SRM and/or SID could be transformed to protein copies/cell and a quantitative model was built to translate MS intensities of all quantified proteins to cellular abundance estimates using the Intensity Based Absolute Quantification (iBAQ) approach [ref 4]."
Comments P.2 left column 2nd paragraph:"Second, [investigators] accurately quantified a subset of identified proteins to establish a 'calibration' for the MS [mass spectrometry] intensities determined for all identified proteins. Here, [they] selected 41 proteins, which [they] expected to be expressed at different abundances. Specifically, [they] selected the enzymes and iso-enzymes of the glycolytic pathway (including proteins with hypothetical function), tricarboxylic acid cycle enzymes and a few other proteins (Supplementary Table 1). These proteins' concentrations were determined in each sample using stable isotope dilution (SID) and selected reaction monitoring (SRM) LC-MS/MS [Liquid chromatography–mass spectrometry] analysis [refs 23, 24] (Supplementary Tables 2 and 3). The concentration range of the 41 proteins covered more than four orders of magnitudes ranging from around 92,000 (Mdh, on acetate medium) to only 2 (YbhA, 3 d into stationary phase) copies per cell."
Entered by Uri M
ID 112273