Although advances in protein labeling methods have made it possible to measure the proteome of mixed cell populations, it has not been possible to isolate cell-type-specific proteomes in vivo. This is because the existing methods for metabolic protein labeling in vivo access all cell types. We report the development of a transgenic mouse line where Cre-recombinase-induced expression of a mutant methionyl-tRNA synthetase (L274G) enables the cell-type-specific labeling of nascent proteins with a non-canonical amino-acid and click chemistry. Using immunoblotting, imaging and mass spectrometry, we use our transgenic mouse to label and analyze proteins in excitatory principal neurons and Purkinje neurons in vitro (brain slices) and in vivo. We discover more than 200 proteins that are differentially regulated in hippocampal excitatory neurons by exposing mice to an environment with enriched sensory cues. Our approach can be used to isolate, analyze and quantitate cell-type-specific proteomes and their dynamics in healthy and diseased tissues.

SummaryHomeostatic scaling adjusts the strength of synaptic connections up or down in response to large changes in input. To identify the landscape of proteomic changes that contribute to opposing forms of homeostatic plasticity, we examined the plasticity-induced changes in the newly synthesized proteome. Cultured rat hippocampal neurons underwent homeostatic up-scaling or down-scaling. We used BONCAT (bio-orthogonal non-canonical amino acid tagging) to metabolically label, capture, and identify newly synthesized proteins, detecting and analyzing 5,940 newly synthesized proteins using mass spectrometry and label-free quantitation. Neither up- nor down-scaling produced changes in the number of different proteins translated. Rather, up- and down-scaling elicited opposing translational regulation of several molecular pathways, producing targeted adjustments in the proteome. We discovered ∼300 differentially regulated proteins involved in neurite outgrowth, axon guidance, filopodia assembly, excitatory synapses, and glutamate receptor complexes. We also identified differentially regulated proteins that are associated with multiple diseases, including schizophrenia, epilepsy, and Parkinson’s disease.

In homeostatic scaling at central synapses, the depth and breadth of cellular mechanisms that detect the offset from the set-point, detect the duration of the offset and implement a cellular response are not well understood. To understand the time-dependent scaling dynamics we treated cultured rat hippocampal cells with either TTX or bicucculline for 2 hr to induce the process of up- or down-scaling, respectively. During the activity manipulation we metabolically labeled newly synthesized proteins using BONCAT. We identified 168 newly synthesized proteins that exhibited significant changes in expression. To obtain a temporal trajectory of the response, we compared the proteins synthesized within 2 hr or 24 hr of the activity manipulation. Surprisingly, there was little overlap in the significantly regulated newly synthesized proteins identified in the early- and integrated late response datasets. There was, however, overlap in the functional categories that are modulated early and late. These data indicate that within protein function groups, different proteomic choices can be made to effect early and late homeostatic responses that detect the duration and polarity of the activity manipulation.