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Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes

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Collagen

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1944))

Abstract

We present the development, optimization, and application of constructs, cell lines, covalent cross-linking methods, and immunoprecipitation strategies that enable robust and accurate determination of collagen interactomes via mass spectrometry-based proteomics. Using collagen type-I as an example, protocols for working with large, repetitive, and GC-rich collagen genes are described, followed by strategies for engineering cells that stably and inducibly express antibody epitope-tagged collagen-I. Detailed steps to optimize collagen interactome cross-linking and perform immunoprecipitations are then presented. We conclude with a discussion of methods to elute collagen interactomes and prepare samples for mass spectrometry-mediated identification of interactors. Throughout, caveats and potential problems researchers may encounter when working with collagen are discussed. We note that the protocols presented herein may be readily adapted to define interactomes of other collagen types, as well as to determine comparative interactomes of normal and disease-causing collagen variants using quantitative isotopic labeling (SILAC)- or isobaric mass tags (iTRAQ or TMT)-based mass spectrometry analysis.

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Acknowledgments

We thank Prof. Joseph Genereux (University of California—Riverside) for technical advice. This work was supported by NIH/NIAMS Grants R03AR067503 and R01AR071443 (to M.D.S.). N.-D.D. was supported by Fonds de Recherche du Québec – Santé (FRQS) and Canadian Institutes of Health Research postdoctoral fellowships. A.S.D. was supported by a NIH Ruth L. Kirschstein predoctoral fellowship (F31AR067615). This work was also supported in part by the NIH/NIEHS under award P30-ES002109 and by a Cancer Center Support (core) Grant P30-CA14051 from the NIH/NCI.

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Authors and Affiliations

  1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA, USA

    Ngoc-Duc Doan, Andrew S. DiChiara & Matthew D. Shoulders

  2. Koch Institute, Massachusetts Institute of Technology, Cambridge, MA, USA

    Amanda M. Del Rosario & Richard P. Schiavoni

Authors
  1. Ngoc-Duc Doan
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  2. Andrew S. DiChiara
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  3. Amanda M. Del Rosario
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  4. Richard P. Schiavoni
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  5. Matthew D. Shoulders
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Corresponding author

Correspondence to Matthew D. Shoulders .

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Editors and Affiliations

  1. Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

    Irit Sagi

  2. Department of Biological Regulation, Weizmann Institute of Science, Rehovot, Israel

    Nikolaos A. Afratis

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Doan, ND., DiChiara, A.S., Del Rosario, A.M., Schiavoni, R.P., Shoulders, M.D. (2019). Mass Spectrometry-Based Proteomics to Define Intracellular Collagen Interactomes. In: Sagi, I., Afratis, N. (eds) Collagen. Methods in Molecular Biology, vol 1944. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-9095-5_7

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  • DOI: https://doi.org/10.1007/978-1-4939-9095-5_7

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-9094-8

  • Online ISBN: 978-1-4939-9095-5

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