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Unbiased Proteomic Study of the Axons of Cultured Rat Cortical Neurons

  • Chih-Fan Chuang
    Chih-Fan Chuang
    Institute of Molecular Medicine, National Tsing Hua University, Hsinchu 30013, Taiwan
    More by Chih-Fan Chuang
    Orcidhttp://orcid.org/0000-0002-7148-6461
  • , 
  • Chih-En King
    Chih-En King
    Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 30013, Taiwan
    More by Chih-En King
  • , 
  • Bo-Wei Ho
    Bo-Wei Ho
    Institute of Systems Neuroscience, National Tsing Hua University, Hsinchu 30013, Taiwan
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  • , 
  • Kun-Yi Chien*
    Kun-Yi Chien
    Department of Biochemistry and Molecular Biology, College of Medicine, Chang Gung University, Taoyuan City 33302, Taiwan
    Clinical Proteomics Core Laboratory, Linkou Chang Gung Memorial Hospital, Taoyuan City 33305, Taiwan
    *E-mail: [email protected]. Phone: 886-3-574-2754.
    More by Kun-Yi Chien
    Orcidhttp://orcid.org/0000-0002-1723-0406
  • , and 
  • Yen-Chung Chang*
    Yen-Chung Chang
    Institute of Systems Neuroscience  and  Department of Life Science, National Tsing Hua University, Hsinchu 30013, Taiwan
    *E-mail: [email protected]. Phone: 886-3-211-8800.
    More by Yen-Chung Chang
Cite this: J. Proteome Res. 2018, 17, 5, 1953–1966
Publication Date (Web):April 10, 2018
https://doi.org/10.1021/acs.jproteome.8b00069
Copyright © 2018 American Chemical Society
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    Abstract

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    The axon is a long projection connecting a neuron to its targets. Here, the axons of cultured rat cortical neurons were isolated with micropatterned chips that enable the separation of axons from their cell bodies. Proteins extracted from isolated axons and whole neurons were subjected to analyses using two-dimensional liquid chromatography–tandem mass spectrometry (2D-LC–MS/MS) analyses without and with stable isotope dimethyl labeling, resulting in the identification of >2500 axonal proteins and 103 axon-enriched proteins. A strong correlation exists between the abundances of axonal proteins and their counterparts in whole neurons. The proteomic results confirm the axonal protein constituents of the subcellular structures documented in earlier electron microscopic studies. Cortical axons have proteins that are components of machineries for protein degradation and the synthesis of soluble, membrane, and secretory proteins, although axons lack conventional Golgi apparatus. Despite the fact that axons lack nucleus, nuclear proteins were identified, and 67 of them were found enriched in axons. Some of the results obtained by the MS-based studies were validated by quantitative Western blotting and immunofluorescence staining analyses. The results represent the first comprehensive description of the axonal protein landscape. The MS proteomics data are available via ProteomeXchange with identifier PXD005527.

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    The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jproteome.8b00069.

    • Antibodies and fluorescent labeling reagents used in current study; proteins detected in culture medium; correlation analyses between abundances of axon proteins and between abundances of whole-cell proteins prepared from two biological replicates; correlation analyses between abundances of axon and whole-cell proteins prepared from the same batch of culture and between abundances of axon and whole-cell proteins prepared from different batches of culture; correlation analyses between A/W ratios calculated from labeling and swap experiments with samples prepared from the same batch of culture and between A/W ratios calculated from labeling and swap experiments with samples prepared from different batches of culture; distribution of A/W ratios obtained in two labeling and two swap experiments (ZIP)

    • Peptides identified in label-free experiments and dimethyl labeling experiments; proteins identified in label-free experiments and dimethyl labeling experiments; lists of proteins identified in axon and whole-cell samples; classification of axonal proteins identified in two independent label-free experiments; classification of 103 proteins consistently enriched in the axon (ZIP)

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