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Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization

Abstract

Macropinocytosis refers to the nonselective uptake of extracellular molecules into many different types of eukaryotic cells within large fluid-filled vesicles named macropinosomes. Macropinosomes are relevant for a wide variety of cellular processes, such as antigen sampling in immune cells, homeostasis in the kidney, cell migration or pathogen uptake. Understanding the molecular composition of the different macropinosomes formed during these processes has helped to differentiate their regulations from other endocytic events. Here, we present a magnetic purification protocol that segregates scarce macropinosomes from other endocytic vesicles at a high purity and in a low-cost and unbiased manner. Our protocol takes advantage of moderate-sized magnetic beads of 100 nm in diameter coupled to mass-spectrometry-based proteomic analysis. Passing the cell lysate through a table-top magnet allows the quick retention of the bead-containing macropinosomes. Unlike other cell-fractionation-based methodologies, our protocol minimizes sample loss and production cost without prerequisite knowledge of the macropinosomes and with minimal laboratory experience. We describe a detailed procedure for the isolation of infection-associated macropinosomes during bacterial invasion and the optimization steps to readily adapt it to various studies. The protocol can be performed in 3 d to provide highly purified and enriched macropinosomes for qualitative proteomic composition analysis.

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Fig. 1: Illustrative overview of the protocol for the magnetic purification of macropinosomes.
Fig. 2: Assembly of the magnetic isolation tubing.
Fig. 3: Overview of the HOKImag magnetic separation system for the purification of macropinosomes.
Fig. 4: Zoom-in of the HOKImag system for the magnetic purification of macropinosomes.
Fig. 5: Retrieval of the magnetic fraction upon magnetic fractionation.
Fig. 6: Expected results from quality controls.
Fig. 7: Expected results from mass spectrometric analysis.

Data availability

Human proteome (Uniprot, v20150113) and Salmonella Typhimurium proteome (Uniprot, 99287) were used in the proteomic analysis. All data included in this protocol are openly available and can be found in the public data online repository PRIDE Archive (PXD012825)30. Source data are provided with this paper. The source data files for Fig. 6b–d are also available at online repository figshare (https://doi.org/10.6084/m9.figshare.14183024 and https://doi.org/10.6084/m9.figshare.14183090, respectively) and in the supporting primary research article30.

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Acknowledgements

The authors thank V. Sohst and N. Reiling (Research Center, Borstel, Germany) for the enthusiasm and great help at the initial stages of this project. Y-Y.C was supported by a fellowship from the Fondation pour la Recherche Médicale (FRM; SPF20160936275). V.S. was supported by a PhD fellowship from the University Paris Diderot (now Université de Paris) allocated by the ENS Paris-Saclay, a grant from the FRM (FRM; FDT20170436843), and an extension grant from the BCI Department of Institut Pasteur. J.E. is a member of the LabEx consortia IBEID and MilieuInterieur. J.E. also acknowledges support from the ANR (grant StopBugEntry and AutoHostPath) and the ERC (CoG EndoSubvert).

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V.S. and Y-Y.C. developed the protocol with input from J.E. V.S. and Y-Y.C. performed the experiments and wrote the manuscript. M.D. and Q.G.G. acquired data, performed analysis and wrote the manuscript. M.M. and J.E. supervised the study, provided feedback on the manuscript and secured funding.

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Correspondence to Virginie Stévenin or Yuen-Yan Chang.

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Peer review information Nature Protocols thanks the anonymous reviewers for their contribution to the peer review of this work.

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Key references using this protocol

Stévenin, V. et al. Cell Rep. 19, 3958–3973.e7 (2019): https://doi.org/10.1016/j.celrep.2019.11.049

Chang, Y. Y. et al. PLoS Pathog. 16, e1008822 (2020): https://doi.org/10.1371/journal.ppat.1008822

Chang, Y. Y. et al. Cell. Microbiol. 23, e13342 (2021): https://doi.org/10.1111/cmi.13342

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Source Data Fig. 6

Unprocessed gel (stained with Coomassie Blue)

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Stévenin, V., Giai Gianetto, Q., Duchateau, M. et al. Purification of infection-associated macropinosomes by magnetic isolation for proteomic characterization. Nat Protoc 16, 5220–5249 (2021). https://doi.org/10.1038/s41596-021-00610-5

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