Efficient, Global-Scale Quantification of Absolute Protein Amounts by Integration of Targeted Mass Spectrometry and Two-Dimensional Gel-Based Proteomics

Abstract

Knowledge on absolute protein concentrations is mandatory for the simulation of biological processes in the context of systems biology. A novel approach for the absolute quantification of proteins at a global scale has been developed and its applicability demonstrated using glucose starvation of the Gram-positive model bacterium Bacillus subtilis and the pathogen Staphylococcus aureus as proof-of-principle examples. Absolute intracellular protein concentrations were initially determined for a preselected set of anchor proteins by employing a targeted mass spectrometric method and isotopically labeled internal standard peptides. Known concentrations of these anchor proteins were then used to calibrate two-dimensional (2-D) gels allowing the calculation of absolute abundance of all detectable proteins on the 2-D gels. Using this approach, concentrations of the majority of metabolic enzymes were determined, and thus a quantification of the players of metabolism was achieved. This new strategy is fast, cost-effective, applicable to any cell type, and thus of value for a broad community of laboratories with experience in 2-D gel-based proteomics and interest in quantitative approaches. Particularly, this approach could also be utilized to quantify existing data sets with the aid of a few standard anchor proteins.

Comprehensive Absolute Quantification of the Cytosolic Proteome of Bacillus subtilis by Data Independent, Parallel Fragmentation in Liquid Chromatography/Mass Spectrometry (LC/MSE)

“…Absolute protein amounts become available by detection and comparison of signal intensities of heavy and light peptides, but only for proteins related to the added synthetic peptides. This method was extended to a more global absolute quantification (AQUA) 1 by calibrating 2D gels with anchor proteins (6). Although the use of internal labeled standards for absolute protein quantification is very precise, availability and costs for such reference peptides are surely limiting.…”

“…Both media contain ammonia salts therefore nitrogen supply is not affected. Cells were harvested during exponential growth (OD 600 nm 0.5, maximal OD 600 nm 4 [condition S] and 4.5 [condition CH]) by centrifugation (8000 ϫ g, 10 min, 4°C) and samples were prepared as described previously (6).…”

“…Absolute QuantificationAbsolute Quantification by AQUA Technique-Absolute quantification by the AQUA method was carried out for six proteins (CitZ, Icd, Mbl, PyrR, SdhA, and Upp) as described previously (6). The selected proteins cover three orders of magnitude from a couple of hundred protein copies per cell to more than ten thousand.…”

Key driving forces in the biosynthesis of autoinducing peptides required for staphylococcal virulence

“…By definition, this process generates the same number of AgrD C fragments in the cytoplasm, which leads to a considerably elevated intracellular concentration because of the much smaller volume of the cytoplasm compared with the medium. For example, AgrD C production would reach an accumulative concentration of 5 mM in 2 h at a rate of 700 nM/s assuming that 1 mL cytoplasm is present in a 1-L cell culture in late exponential phase (24). Constant and efficient AIP production, therefore, necessitates simultaneous removal of AgrD C , because as we have shown, elevating the levels of this fragment can perturb the equilibrium of AgrB-mediated proteolytic cyclization (Fig.…”

An ancient family of mobile genomic islands introducing cephalosporinase and carbapenemase genes in Enterobacteriaceae

“…For sample preparation, cells were collected in the mid-exponential and stationary growth phases by centrifugation and disrupted by bead-beating with glass beads (~ 0.1 mm diameter) in a Precellys 24 homogenisator (Bertin Technologies, France) as described previously [49,50]. Glass beads and cell debris were removed by centrifugation (21,000 × g, 10 min, 4°C).…”