JournalNeural Excitability, Synapses, and Glia

Cell-type-specific metabolic labeling, detectionand identification of nascent proteomes in vivo

Workflow for cell-type-specific protein labeling and purification.Workflow for protein labeling in mice by adding ANL to the drinking water or by intraperitoneal injection. The tissue under study is collected and the proteins containing ANL are clicked (BONCAT); then the labeled proteins are isolated by affinity purification and eluted by cleaving the alkyne. Purified proteins are identified by MS. The corresponding steps from the protocol are indicated.

A big challenge in proteomics is the identification of cell-type-specific proteomes in vivo. This protocol describes how to label, purify and identify cell-type-specific proteomes in living mice. To make this possible, we created a Cre-recombinase-inducible mouse line expressing a mutant methionyl-tRNA synthetase (L274G), which enables the labeling of nascent proteins with the non-canonical amino acid azidonorleucine (ANL). This amino acid can be conjugated to different affinity tags by click chemistry. After affinity purification (AP), the labeled proteins can be identified by tandem mass spectrometry (MS/MS). With this method, it is possible to identify cell-type-specific proteomes derived from living animals, which was not possible with any previously published method. The reduction in sample complexity achieved by this protocol allows for the detection of subtle changes in cell-type-specific protein content in response to environmental changes. This protocol can be completed in ~10 d (plus the time needed to generate the mouse lines, the desired labeling period and MS analysis).

Alvarez-Castelao, B., Schanzenbaecher, C.T., Langer, J.D., and Schuman, E.M. (2019). Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo. Nature Protocols, published online on January 4, 2019.