Procedures for the biochemical enrichment and proteomic analysis of the cytoskeletome

Sunkyu Choi, Jonathan Kelber, Xinning Jiang, Jan Strnadel, Ken Fujimura, Martina Pasillas, Judith Coppinger, Richard Klemke

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3 Citations (Scopus)

Abstract

The cell cytoskeleton is composed of microtubules, intermediate filaments, and actin that provide a rigid support structure important for cell shape. However, it is also a dynamic signaling scaffold that receives and transmits complex mechanosensing stimuli that regulate normal physiological and aberrant pathophysiological processes. Studying cytoskeletal functions in the cytoskeleton's native state is inherently difficult due to its rigid and insoluble nature. This has severely limited detailed proteomic analyses of the complex protein networks that regulate the cytoskeleton. Here, we describe a purification method that enriches for the cytoskeleton and its associated proteins in their native state that is also compatible with current mass spectrometry-based protein detection methods. This method can be used for biochemical, fluorescence, and large-scale proteomic analyses of numerous cell types. Using this approach, 2346 proteins were identified in the cytoskeletal fraction of purified mouse embryonic fibroblasts, of which 635 proteins were either known cytoskeleton proteins or cytoskeleton-interacting proteins. Functional annotation and network analyses using the Ingenuity Knowledge Database of the cytoskeletome revealed important nodes of interconnectivity surrounding well-established regulators of the actin cytoskeleton and focal adhesion complexes. This improved cytoskeleton purification method will aid our understanding of how the cytoskeleton controls normal and diseased cell functions.

Original languageEnglish
Pages (from-to)102-107
Number of pages6
JournalAnalytical Biochemistry
Volume446
Issue number1
DOIs
Publication statusPublished - 1 Feb 2014

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Keywords

  • Actin scaffold
  • Cytoskeleton
  • Focal adhesion
  • Proteomics

ASJC Scopus subject areas

  • Biophysics
  • Biochemistry
  • Molecular Biology
  • Cell Biology

Cite this

Choi, S., Kelber, J., Jiang, X., Strnadel, J., Fujimura, K., Pasillas, M., Coppinger, J., & Klemke, R. (2014). Procedures for the biochemical enrichment and proteomic analysis of the cytoskeletome. Analytical Biochemistry, 446(1), 102-107. https://doi.org/10.1016/j.ab.2013.10.025