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Shotgun Proteomics pp 25–45Cite as

Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 2259))

Abstract

Laser capture microdissection (LCM) provides a fast, specific, and versatile method to isolate and enrich cells in mixed populations and/or subcellular structures, for further proteomic study. Furthermore, mass spectrometry (MS) can quickly and accurately generate differential protein expression profiles from small amounts of samples. Although cellular protrusions—such as tunneling nanotubes, filopodia, growth cones, invadopodia, etc.—are involved in essential physiological and pathological actions such as phagocytosis or cancer-cell invasion, the study of their protein composition is progressing slowly due to their fragility and transient nature. The method described herein, combining LCM and MS, has been designed to identify the proteome of different cellular protrusions. First, cells are fixed with a novel fixative method to preserve the cellular protrusions, which are isolated by LCM. Next, the extraction of proteins from the enriched sample is optimized to de-crosslink the fixative agent to improve the identification of proteins by MS. The efficient protein recovery and high sample quality of this method enable the protein profiling of these small and diverse subcellular structures.

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Acknowledgments

This work was supported by a 2014 CSUPERB New Investigator Grant and the National Institute of General Medical Sciences of the National Institutes of Health under Award Number SC2GM111144 awarded to K.G.

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Author notes

  1. Ana Gordon

    Present address: Department of Cell Biology, Physiology, and Immunology, University of Córdoba, Córdoba, Spain

Authors and Affiliations

  1. Biology Department, California State University Fresno, Fresno, CA, USA

    Ana Gordon & Karine Gousset

Authors
  1. Ana Gordon
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  2. Karine Gousset
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Corresponding author

Correspondence to Karine Gousset .

Editor information

Editors and Affiliations

  1. Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain

    Mónica Carrera

  2. Department of Food Technology, Institute of Marine Research (IIM), Spanish National Research Council (CSIC), Vigo, Spain

    Jesús Mateos

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Gordon, A., Gousset, K. (2021). Utilization of Laser Capture Microdissection Coupled to Mass Spectrometry to Uncover the Proteome of Cellular Protrusions. In: Carrera, M., Mateos, J. (eds) Shotgun Proteomics. Methods in Molecular Biology, vol 2259. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1178-4_3

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  • DOI: https://doi.org/10.1007/978-1-0716-1178-4_3

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1177-7

  • Online ISBN: 978-1-0716-1178-4

  • eBook Packages: Springer Protocols

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