Proteome-wide absolute quantification using a generic internal standard

Absolute (molar) quantification of proteins helps us to determine stoichiometric ratios within metabolic pathways. Neither genome nor transcriptome sheds light on how metabolic networks are organized: genomics tells what could be the components; transcriptomics shows which genes are expressed, but neither of the two translate into the actual protein concentrations. Here, we aimed at demonstrating that proteome-wide absolute quantification supports the direct comparison of proteome composition of phylogenetically distant minimal organisms. These minimal organisms help us to understand the fundamental principles of life and the basic requirements for cellular function, including metabolism. We aim to determine and compare the quantities of functionally related proteins in the whole cells of minimal JCVI-syn3A (2) bacteria and near minimal bacterium Mesoplasma Florium (3). We underscore that protein levels comparison could not be achieved by considering their genomes or (if available) transcriptomes.

We have developed a workflow for proteome –wide absolute quantification by DDA with a family of FUGIS (Fully Unlabelled Generic Internal Standard) (1) proteins as the internal standard. The FUGIS chimeric protein is co-digested with a known amount of BSA (reference protein) along with protein test samples. The digest is analyzed using LC-MS/MS workflow and the raw data files are processed with MSFragger (Fig 1A). The .tsv output from the MSFragger with peptide ID"s and their respective raw XIC peak areas is used by in-house built GlobeQuant(1) software for quantification. The software calculates the molar amount of the FUGIS peptides and relates it to the median area of XIC peaks of FUGIS peptides peaks and further uses it as a single-point calibrant to determine the molar abundance of any codigested protein. The peptides of the codigested sample proteins are selected by the principle of BestNAverage, where the "BestN" is selected from the Top peptides based on a criteria of CV% (Fig 1B).

These minimal cell organisms allow us to perform absolute quantification in an untargeted way to quantify the whole proteome across organisms hence quantifying and comparing the homologous proteins at the organism level.

References

J. Proteome Res.2022, 21, 1, 132–141. doi:10.1021/acs.jproteome.1c00596 Pelletier, J.F. et al. (2021) "Genetic requirements for cell division in a genomically minimal cell", Cell, 184(9). doi:10.1016/j.cell.2021.03.008. Matteau, D. et al. (2020) "Integrative characterization of the near‐minimal bacterium "mesoplasma florum", Molecular Systems Biology, 16(12). doi:10.15252/msb.20209844.