Taking advantage of ideas for advancing data acquisition and interpretation, we developed a next generation methods that increase the sensitivity, data completeness and flexibility of single-cell protein analysis. These allow prioritization of thousands of proteins and highly parallel analysis of both single-cells and peptides. All of these methods can be implemented using accessible commercial equipment.
All living cells must coordinate their metabolism, growth, division, and differentiation with their gene expression. Gene expression is regulated at multiple layers, from histone modifications (histone code) through RNA processing to protein degradation. While most layers are extensively studied, the regulatory role of specialized ribosomes (ribosome code) is largely unexplored. Such specialization has been suggested by the differential transcription of ribosomal proteins (RPs)and by the observation that mutations of RPs have highly specific phenotypes; particular RP mutations can cause diseases, such as cancer and Diamond Blackfan anemia, and affect selectively the synthesis of some proteins but not of others. This selectivity and the differential RP transcription raise the hypothesis that cells may build specialized ribosomes with different stoichiometries among RPs as a means of regulating protein synthesis.
While the existence of specialized ribosomes has been hypothesized for decades, experimental and analytical roadblocks (such as the need for accurate quantification of homologous proteins and their modifications) have limited the evidence to only a few examples, e.g., the phosphorylation of RP S6. We developed methods to clear these roadblocks and obtained direct evidence for differential stoichiometry among core RPs in unperturbed yeast and mammalian stem cells and its fitness phenotypes. We aim to characterize ribosome specialization and its coordination with gene regulation, metabolism, and cell growth and differentiation. We want to understand quantitatively, conceptually, and mechanistically this coordination with emphasis on direct precision measurements of metabolic fluxes, protein synthesis and degradation rates in absolute units, molecules per cell per hour.