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Nat Protoc. 2019 Feb;14(2):556-575. doi: 10.1038/s41596-018-0106-6.

Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo.

Author information

1
Max Planck Institute for Brain Research, Frankfurt, Germany. beatriz.alvarez-castelao@brain.mpg.de.
2
Max Planck Institute for Brain Research, Frankfurt, Germany.
3
Max Planck Institute of Biophysics, Frankfurt, Germany.
4
Max Planck Institute for Brain Research, Frankfurt, Germany. erin.schuman@brain.mpg.de.

Abstract

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).

PMID:
30610240
DOI:
10.1038/s41596-018-0106-6
[Indexed for MEDLINE]

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