Cited by 84Advanced Proteomics Techniques and ApplicationsClick Chemistry and ApplicationsBiotin and Related Studies

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

Alvarez-Castelao, Beatriz, Schanzenbächer, Christoph T., Langer, Julian D., Schuman, Erin M.·2019·Nature Protocols

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).

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Crossref · 10.1038/s41596-018-0106-6

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Authors
Alvarez-Castelao, B., Schanzenbächer, C. T., Langer, J. D., & Schuman, E. M.
Affiliations
  • Max Planck Institute of Biophysics (DE)
  • Max Planck Institute for Brain Research (DE)
Corresponding author
Schuman, Erin M.
Venue
Nature Protocols
Published
2019-01-04
Volume
14
Issue
2
Pages
556-575
ISSN
  • 1750-2799 (ISSN-L)
  • 1754-2189
OpenAlex
W2908417496
Cited by
84
References
38
Citations by year
2026: 6 · 2025: 11 · 2024: 16 · 2023: 11 · 2022: 8 · 2021: 13 · 2020: 16 · 2019: 3
FWCI
3.99
Type
journal article
Language
en
Open access
closed
Locations
  • Nature Protocols (publishedVersion)
  • PubMed (publishedVersion)
  • MPG.PuRe (Max Planck Society) (submittedVersion)
Topics
  • Physical Sciences › Chemistry › Spectroscopy › Advanced Proteomics Techniques and Applications
  • Physical Sciences › Chemistry › Organic Chemistry › Click Chemistry and Applications
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cell Biology › Biotin and Related Studies
Keywords
nascent protein, metabolic labeling, click chemistry, affinity purification, tandem mass spectrometry
MeSH terms
Tandem Affinity Purification, Animals, Azides, Methionine-tRNA Ligase, Mice, Transgenic, Mutation, Norleucine, Organ Specificity, Staining and Labeling, Gene Expression, Integrases, Proteome, Proteomics, Mice, Tandem Mass Spectrometry, Click Chemistry

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