Cell-type-specific metabolic labeling, detection and identification of nascent proteomes in vivo
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).
Integrity signals
Registry
Cites
Statistics
Claims the paper makes
Claims other papers make about this one
Each is checked against this paper’s own text: does it say what the citing paper says it does?
BONCAT was utilized to characterize excitatory and inhibitory neuronal proteomes in adult mice.
“Recently, BONCAT was utilized to characterize excitatory and inhibitory neuronal proteomes in adult mice [158, 159].”
From Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau (2021) · cites this paper as [159]
Cites
Wessel, D., & Flügge, U. (1984) · Analytical Biochemistry · doi:10.1016/0003-2697(84)90782-6 · Cited by 3,997 · see citing works
SUnSET, a nonradioactive method to monitor protein synthesis
Schmidt, E. K., Clavarino, G., Ceppi, M., & Pierre, P. (2009) · Nature Methods · doi:10.1038/nmeth.1314 · Cited by 1,706 · see citing works
In Vivo Imaging of Membrane-Associated Glycans in Developing Zebrafish
Laughlin, S. T., Baskin, J. M., Amacher, S. L., & Bertozzi, C. R. (2008) · Science · doi:10.1126/science.1155106 · Cited by 1,031 · see citing works
Dieterich, D. C., Link, A. J., Graumann, J., Tirrell, D. A., & Schuman, E. M. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0601637103 · Cited by 874 · see citing works
Self-citationClick Chemistry in Complex Mixtures: Bioorthogonal Bioconjugation
McKay, C. S., & Finn, M. G. (2014) · Chemistry & Biology · doi:10.1016/j.chembiol.2014.09.002 · Cited by 771 · see citing works
Chemical and Anatomical Plasticity of Brain
Bennett, E. L., Diamond, M. C., Krech, D., & Rosenzweig, M. R. (1964) · Science · doi:10.1126/science.146.3644.610 · Cited by 767 · see citing works
In situ visualization and dynamics of newly synthesized proteins in rat hippocampal neurons
Dieterich, D. C., Hodas, J. J. L., Gouzer, G., Shadrin, I. Y., Ngo, J. T., Triller, A., Tirrell, D. A., & Schuman, E. M. (2010) · Nature Neuroscience · doi:10.1038/nn.2580 · Cited by 470 · see citing works
Self-citationE unum pluribus: multiple proteins from a self-processing polyprotein
de Felipe, P., Luke, G. A., Hughes, L. E., Gani, D., Halpin, C., & Ryan, M. D. (2005) · Trends in biotechnology · doi:10.1016/j.tibtech.2005.12.006 · Cited by 358 · see citing works
Fornasiero, E. F., Mandad, S., Wildhagen, H., Alevra, M., Rammner, B., Keihani, S., Opazo, F., Urban, I., Ischebeck, T., Sakib, M. S., et al. (2018) · Nature Communications · doi:10.1038/s41467-018-06519-0 · Cited by 340 · see citing works
Gillet, L. C., Leitner, A., & Aebersold, R. (2016) · Annual Review of Analytical Chemistry · doi:10.1146/annurev-anchem-071015-041535 · Cited by 323 · see citing works
Direct visualization of newly synthesized target proteins in situ
Dieck, S. T., Kochen, L., Hanus, C., Heumüller, M., Bartnik, I., Nassim-Assir, B., Merk, K., Mosler, T., Garg, S., Bunse, S., et al. (2015) · Nature Methods · doi:10.1038/nmeth.3319 · Cited by 294 · see citing works
Self-citationKinetic Analysis of Protein Stability Reveals Age-Dependent Degradation
McShane, E., Sin, C., Zauber, H., Wells, J. N., Donnelly, N., Wang, X., Hou, J., Chen, W., Štorchová, Z., Marsh, J. A., et al. (2016) · Cell · doi:10.1016/j.cell.2016.09.015 · Cited by 291 · see citing works
Feist, P. E., & Hummon, A. B. (2015) · International Journal of Molecular Sciences · doi:10.3390/ijms16023537 · Cited by 289 · see citing works
Measuring Protein Synthesis With SUnSET: A Valid Alternative to Traditional Techniques?
Goodman, C. A., & Hornberger, T. A. (2013) · Exercise and Sport Sciences Reviews · doi:10.1097/jes.0b013e3182798a95 · Cited by 233 · see citing works
Cell-type-specific metabolic labeling of nascent proteomes in vivo
Alvarez-Castelao, B., Schanzenbächer, C. T., Hanus, C., Glock, C., tom Dieck, S., Dörrbaum, A. R., Bartnik, I., Nassim-Assir, B., Ciirdaeva, E., Mueller, A., et al. (2017) · Nature Biotechnology · doi:10.1038/nbt.4016 · Cited by 219 · see citing works
Self-citationCleavable Biotin Probes for Labeling of Biomolecules via Azide−Alkyne Cycloaddition
Szychowski, J., Mahdavi, A., Hodas, J. J. L., Bagert, J. D., Ngo, J. T., Landgraf, P., Dieterich, D. C., Schuman, E. M., & Tirrell, D. A. (2010) · Journal of the American Chemical Society · doi:10.1021/ja1083909 · Cited by 217 · see citing works
Self-citationCell-selective metabolic labeling of proteins
Ngo, J. T., Champion, J. A., Mahdavi, A., Tanrikulu, I. C., Beatty, K. E., Connor, R. E., Yoo, T. H., Dieterich, D. C., Schuman, E. M., & Tirrell, D. A. (2009) · Nature Chemical Biology · doi:10.1038/nchembio.200 · Cited by 194 · see citing works
Self-citationLink, A. J., Vink, M. K. S., Agard, N. J., Prescher, J. A., Bertozzi, C. R., & Tirrell, D. A. (2006) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.0601167103 · Cited by 189 · see citing works
Genetic code expansion in stable cell lines enables encoded chromatin modification
Elsässer, S. J., Ernst, R. J., Walker, O. S., & Chin, J. W. (2016) · Nature Methods · doi:10.1038/nmeth.3701 · Cited by 164 · see citing works
Nascent Proteome Remodeling following Homeostatic Scaling at Hippocampal Synapses
Schanzenbächer, C. T., Sambandan, S., Langer, J. D., & Schuman, E. M. (2016) · Neuron · doi:10.1016/j.neuron.2016.09.058 · Cited by 137 · see citing works
Self-citationThe Regulation of Synaptic Protein Turnover
Alvarez-Castelao, B., & Schuman, E. M. (2015) · Journal of Biological Chemistry · doi:10.1074/jbc.r115.657130 · Cited by 110 · see citing works
Self-citationCell-specific proteomic analysis in Caenorhabditis elegans
Yuet, K. P., Doma, M. K., Ngo, J. T., Sweredoski, M. J., Graham, R. L. J., Moradian, A., Hess, S. Y., Schuman, E. M., Sternberg, P. W., & Tirrell, D. A. (2015) · Proceedings of the National Academy of Sciences · doi:10.1073/pnas.1421567112 · Cited by 100 · see citing works
Self-citationIn Vivo Quantitative Proteomics: The SILAC Mouse
Zanivan, S. R., Krüger, M., & Mann, M. (2011) · Methods in molecular biology · doi:10.1007/978-1-61779-166-6_25 · Cited by 98 · see citing works
A General Approach to Detect Protein Expression In Vivo Using Fluorescent Puromycin Conjugates
Starck, S. R., Green, H. M., Alberola‐Ila, J., & Roberts, R. W. (2004) · Chemistry & Biology · doi:10.1016/j.chembiol.2004.05.011 · Cited by 87 · see citing works
Landgraf, P., Antileo, E. R., Schuman, E. M., & Dieterich, D. C. (2015) · Methods in Molecular Biology · doi:10.1007/978-1-4939-2272-7_14 · Cited by 78 · see citing works
Self-citationEngineered Aminoacyl-tRNA Synthetase for Cell-Selective Analysis of Mammalian Protein Synthesis
Mahdavi, A., Hamblin, G. D., Jindal, G. A., Bagert, J. D., Dong, C., Sweredoski, M. J., Hess, S. Y., Schuman, E. M., & Tirrell, D. A. (2016) · Journal of the American Chemical Society · doi:10.1021/jacs.5b08980 · Cited by 74 · see citing works
Self-citationMcClatchy, D. B., Ma, Y., Liu, C., Stein, B. D., Martínez‐Bartolomé, S., Vasquez, D. S., Hellberg, K., Shaw, R. J., & Yates, J. R. (2015) · Journal of Proteome Research · doi:10.1021/acs.jproteome.5b00653 · Cited by 74 · see citing works
Woodruff‐Pak, D. S. (2006) · Neuroscience · doi:10.1016/j.neuroscience.2006.03.070 · Cited by 66 · see citing works
Schanzenbächer, C. T., Langer, J. D., & Schuman, E. M. (2018) · eLife · doi:10.7554/elife.33322 · Cited by 62 · see citing works
Self-citationGauthier, N. P., Soufi, B., Walkowicz, W. E., Pedicord, V. A., Mavrakis, K. J., Maček, B., Gin, D. Y., Sander, C., & Miller, M. L. (2013) · Nature Methods · doi:10.1038/nmeth.2529 · Cited by 61 · see citing works
Application of bio-orthogonal proteome labeling to cell transplantation and heterochronic parabiosis
Liu, Y., Conboy, M. J., Mehdipour, M., Liu, Y., Tran, T. P., Blotnick, A. M., Rajan, P., Santos, T. C., & Conboy, I. M. (2017) · Nature Communications · doi:10.1038/s41467-017-00698-y · Cited by 60 · see citing works
3 The Medicinal Chemistry of the Azido Group
Griffin, R. J. (1994) · Progress in medicinal chemistry · doi:10.1016/s0079-6468(08)70020-1 · Cited by 59 · see citing works
Barrett, R. M., Liu, H.-W., Jin, H., Goodman, R. H., & Cohen, M. S. (2016) · ACS Chemical Biology · doi:10.1021/acschembio.5b01076 · Cited by 46 · see citing works
Puromycin labeling does not allow protein synthesis to be measured in energy-starved cells
Marciano, R., Leprivier, G., & Rotblat, B. (2018) · Cell Death and Disease · doi:10.1038/s41419-017-0056-x · Cited by 38 · see citing works
Link, A. J., Vink, M. K. S., & Tirrell, D. A. (2007) · Nature Protocols · doi:10.1038/nprot.2007.268 · Cited by 34 · see citing works
Du, S., Wang, D., Lee, J., Peng, B., Ge, J., & Yao, S. Q. (2017) · Chemical Communications · doi:10.1039/c7cc04536k · Cited by 22 · see citing works
Pulse-Chase Labeling Techniques for the Analysis of Protein Maturation and Degradation
Jansens, A., & Braakman, I. (2003) · Humana Press eBooks · doi:10.1385/1-59259-394-1:133 · Cited by 19 · see citing works
Liu, Y., Conboy, M. J., Mehdipour, M., Liu, Y., Tran, T. P., Blotnick, A. M., Rajan, P., Santos, T. C., & Conboy, I. M. (2018) · Nature Communications · doi:10.1038/s41467-017-02779-4 · Cited by 1 · see citing works
Flagged first, then by citations
Cited by
Sorted by citations
Early Alzheimer’s disease pathology in human cortex involves transient cell states
Gazestani, V., Kamath, T., Nadaf, N. M., Dougalis, A., Burris, S. J., Rooney, B., Junkkari, A., Vanderburg, C., Pelkonen, A., Gomez-Budia, M., et al. (2023) · Cell · doi:10.1016/j.cell.2023.08.005 · Cited by 209 · see citing works
Systems-based proteomics to resolve the biology of Alzheimer’s disease beyond amyloid and tau
Rayaprolu, S., Higginbotham, L., Bagchi, P., Watson, C. M., Zhang, T., Levey, A. I., Rangaraju, S., & Seyfried, N. T. (2021) · Neuropsychopharmacology · doi:10.1038/s41386-020-00840-3 · Cited by 118 · see citing works
- 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
- Licence
- www.springer.com/tdm (tdm)
- 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
About these checks
Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.
Statistics and arithmetic are recomputed, not judged.
Confidence bands are shown only for fields calibrated on our annotated test set. None is calibrated yet, so no band is shown.
What counts as a claim ›How we measure confidence ›How we check this ›
Are you an author, or do you see a problem with this page? Request a review