Life science research faces an increasing demand for absolute quantification of biomolecules to determine the molecular composition of a cell and to support mathematical modelling of biochemical processes. Here, we implemented a universally applicable, label-free strategy to estimate absolute cellular protein concentrations on a proteome-wide scale based on SWATH mass spectrometry. We applied this strategy to study proteomic reorganisation in the human pathogen Mycobacterium tuberculosis during exponential growth, hypoxia-induced dormancy and resuscitation. The resulting data set covering >2000 proteins reveals how protein biomass is distributed among cellular functions and dynamically remodelled in response to hypoxic stress. We found that the DosR regulon contributes 20% to the entire cellular protein content during dormancy, whereas the fraction of ribosomal proteins remains largely unchanged at 5-7%. Knowledge of protein copies/cell furthermore allowed us to translate effects of protein regulation into changes in maximal enzymatic reaction velocities, enhancing our understanding of metabolic adaptations.

To ensure accurate absolute quantitation results, the linear dynamic quantification range and the lower limit of quantification for each reference peptide was determined by performing a dilution series experiment. In the end, 51 peptides showed good linear response (slope close to 1, axis intercept close to 0, R2 > 0.94) and suitable lower limit of quantification with respect to endogenous protein levels.