Proteome Birthdating Reveals Age-Selectivity of Protein Ubiquitination

Within a cell, proteins have distinct and highly variable half-lives. As a result, the molecular ages of proteins can range from seconds to years. How the age of a protein influences its environmental interactions is a largely unexplored area of biology. To investigate the age-selectivity of cellular pathways, we developed a methodology termed “proteome birthdating” that barcodes proteins based on their time of synthesis. We demonstrate that this approach provides accurate measurements of protein turnover kinetics from a single biological sample encoding multiple labeling time-points. As a first application of the birthdated proteome, we investigated the age distribution of the human ubiquitinome. Our results indicate that the vast majority of ubiquitinated proteins in a cell consist of newly synthesized proteins and that these young proteins constitute the bulk of the degradative flux through the proteasome. Rapidly ubiquitinated nascent proteins are enriched in cytosolic subunits of large protein complexes. Conversely, proteins destined for the secretory pathway and vesicular transport have older ubiquitinated populations. Our data also identify a smaller subset of older ubiquitinated cellular proteins that do not appear to be targeted to the proteasome for rapid degradation. Together, our data provide an age census of the human ubiquitinome and establish proteome birthdating as a robust methodology for investigating the protein age-selectivity of diverse cellular pathways. [Display omitted] •Proteome birthdating barcodes the proteome based on protein age.•Age-selectivity of cellular pathways can be investigated.•Proteome turnover dynamics can be analyzed within a single biological sample.•An age census of the human ubiquitinome was conducted.•Ubiquitinated newly synthesized proteins are the major substrates for the proteasome. Cellular proteins have different ages - whereas some have been recently synthesized, others have existed in the cell for days. How a protein’s age influences its function is difficult to study. We developed a method named “proteome birthdating” that partitions proteins into discernible age groups. We examined the age-selectivity of ubiquitination and show that proteasomal targets consist mostly of young proteins. Proteome birthdating is useful for analyzing proteome turnover dynamics and investigating the functional consequences of protein age.