Cell type-specific in vivo proteomes with a multicopy mutant methionyl tRNA synthetase mouse line

The functional diversity of cells is driven by the different proteins they express. While improvements in protein labeling techniques have enabled the measurement of proteomes with increased sensitivity, measuring cell type-specific proteomes in vivo remains challenging. One of the most useful pipelines is bio-orthogonal noncanonical amino acid tagging (BONCAT) with the MetRS* system, consisting of a transgenic mouse line expressing a mutant methionyl-tRNA synthetase (MetRS*) controlled by Cre recombinase expression. This system allows cell type-specific labeling of proteins with a noncanonical amino acid (azidonorleucine, ANL), which can be subsequently conjugated to affinity or fluorescent tags using click chemistry. Click-modified proteins can then be visualized, purified and identified. The reduction in sample complexity enables the detection of small changes in protein composition. Here, we describe a multicopy MetRS* mouse line (3xMetRS* mouse line) that exhibits markedly enhanced ANL protein labeling, boosting the sensitivity and temporal resolution of the system and eliminating the need for working under methionine depletion conditions. Cell type-specific in vivo labeling is possible even in heterozygous animals, thus offering an enormous advantage for crossing the line into mutation and disease-specific backgrounds. Using the 3xMetRS* line, we identified the in vivo proteome of a sparse cell population-the dopaminergic neurons of the olfactory bulb-and furthermore determined newly synthesized proteins after short labeling durations following a single intraperitoneal ANL injection.