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Cell-type-specific quantification of protein synthesis in vivo

Nature Protocols volume 14, pages 441–460 (2019)Cite this article

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Abstract

Although protein synthesis is a conserved and essential cellular function, it is often regulated in a cell-type-specific manner to influence cell fate, growth and homeostasis. Most methods used to measure protein synthesis depend on metabolically labeling large numbers of cells with radiolabeled amino acids or amino acid analogs. Because these methods typically depend on specialized growth conditions, they have been largely restricted to yeast, bacteria and cell lines. Application of these techniques to investigating protein synthesis within mammalian systems in vivo has been challenging. The synthesis of O-propargyl-puromycin (OP-Puro), an analog of puromycin that contains a terminal alkyne group, has facilitated the quantification of protein synthesis within individual cells in vivo. OP-Puro enters the acceptor site of ribosomes and incorporates into nascent polypeptide chains. Incorporated OP-Puro can be detected through a click-chemistry reaction that links it to a fluorescently tagged azide molecule. In this protocol, we describe how to administer OP-Puro to mice, obtain cells of interest (here, we use bone marrow cells) just 1 h later, and quantify the amount of protein synthesized per hour by flow cytometry on the basis of OP-Puro incorporation. We have used this approach to show that hematopoietic stem cells (HSCs) exhibit an unusually low rate of protein synthesis relative to other hematopoietic cells, and it can be easily adapted to quantify cell-type-specific rates of protein synthesis across diverse mammalian tissues in vivo. Measurement of protein synthesis within bone marrow cells in a cohort of six mice can be achieved in 8–10 h.

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Fig. 1: Molecular structure of OP-Puro.
Fig. 2: Overview of key steps in the procedure to measure protein synthesis in vivo on the basis of OP-Puro incorporation.
Fig. 3: Obtaining bone marrow cells from mouse long bones.
Fig. 4: HSCs synthesize less protein per hour than unfractionated bone marrow cells.
Fig. 5: Restricted myeloid progenitors have much higher protein synthesis rates than HSCs.
Fig. 6: OP-Puro incorporation by HSCs and myeloid progenitor cells in vivo.

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Data availability

The datasets generated during and/or analyzed during the current study are available from the corresponding author upon reasonable request.

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Acknowledgements

This work was supported by the NIDDK (R01DK116951), the V Foundation for Cancer Research, the American Society of Hematology, the Sanford Stem Cell Clinical Center, a Chancellor’s Research Excellence Scholarship and an NCI Cancer Center Support Grant to the Moores Cancer Center (P30-CA 023100). Flow cytometry was performed at the Flow Cytometry Core Facility at the La Jolla Institute for Allergy and Immunology. The FACSAria II cell sorter was acquired through the Shared Instrumentation Grant Program (S10 RR027366).

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Authors and Affiliations

  1. Division of Regenerative Medicine, Department of Medicine, Moores Cancer Center, University of California San Diego, La Jolla, CA, USA

    Lorena Hidalgo San Jose & Robert A. J. Signer

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  1. Lorena Hidalgo San Jose
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Contributions

R.A.J.S. developed the original protocol. L.H.S.J. performed the experiments and generated the data shown. L.H.S.J. and R.A.J.S. wrote the manuscript.

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Correspondence to Robert A. J. Signer.

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Key references using this protocol

Signer, R. A. J., Magee, J. A., Salic, A. & Morrison, S. J. Nature 509, 49–54 (2014): https://www.nature.com/articles/nature13035

Signer, R. A. J. et al. Genes Dev. 30, 1698–1703 (2016): http://genesdev.cshlp.org/content/30/15/1698

Integrated supplementary information

Supplementary Figure 1 Comparison between OP-Puro and anti-puromycin antibody-mediated detection of protein synthesis within mouse bone marrow cells in vivo.

a, MFI of OP-Puro (black) and Puromycin (blue) in unfractionated bone marrow cells, HSCs, CMPs, GMPs, and MEPs (n = 3 for each method). Background fluorescence has been subtracted from each population. Data represent mean ± standard deviation. Statistical significance between methods within each cell type was assessed by Student’s t-test.; ***P<0.001. b, Representative histograms show OP-Puro incorporation (red) and puromycin incorporation (blue) in unfractionated bone marrow cells compared to unfractionated bone marrow cells from a PBS injected control mouse (gray). All procedures in this protocol involving mice were approved by the UC San Diego Institutional Animal Care and Use Committee.

Supplementary Figure 2 The SUnSET method cannot be applied to measure protein synthesis in vivo.

A representative histogram shows the inability to detect puromycin on the cell surface of unfractionated bone marrow cells (blue). Unfractionated bone marrow cells from a PBS injected control mouse are shown in gray. All procedures in this protocol involving mice were approved by the UC San Diego Institutional Animal Care and Use Committee.

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Hidalgo San Jose, L., Signer, R.A.J. Cell-type-specific quantification of protein synthesis in vivo. Nat Protoc 14, 441–460 (2019). https://doi.org/10.1038/s41596-018-0100-z

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