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Quantitative Phosphoproteomic Analysis of Brain Tissues

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Book cover Neuroproteomics

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1598))

Abstract

Protein phosphorylation regulates brain development and neuronal activities; and dysregulation of phosphorylation contributes to neurobiological disorders. Phosphoproteomic analysis provides comprehensive modification maps for measuring protein activities in cellular pathways and biological processes. Here, we introduce a mass spectrometry (MS)-based protocol to quantitatively analyze the phosphoproteome of human postmortem brains of Alzheimer’s disease. In this isobaric labeling protocol, up to ten brain samples are selected from control and diseased cases for comparison. Approximately 1 mg proteins per sample are extracted, digested, labeled, and then mixed at an equal ratio. To improve the coverage of phosphoproteome, the peptide mix is further fractionated by offline basic pH reversed-phase liquid chromatography (LC) with high-resolution power. Phosphopeptides in each fraction are then enriched by the titanium dioxide method and analyzed by online acidic pH reverse phase LC-MS/MS, leading to the analysis of tens of thousands of phosphorylation events. This protocol can also be adapted to profile phosphoproteome in other biological samples.

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Acknowledgments

This work was partially supported by the National Institutes of Health (R01GM114260, R01AG047928, and R01AG053987), the American Cancer Society (RSG-09-181), and ALSAC (American Lebanese Syrian Associated Charities).

Author information

Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children’s Research Hospital, MS 311, D1034C, 262 Danny Thomas Place, Memphis, TN, 38105-3678, USA

    Bing Bai & Junmin Peng

  2. Department of Developmental Neurobiology, St. Jude Children’s Research Hospital, MS 311, D1034C, 262 Danny Thomas Place, Memphis, TN, 38105-3678, USA

    Bing Bai & Junmin Peng

  3. St. Jude Proteomics Facility, St. Jude Children’s Research Hospital, MS 311, D1034C, 262 Danny Thomas Place, Memphis, TN, 38105-3678, USA

    Haiyan Tan & Junmin Peng

Authors
  1. Bing Bai
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  2. Haiyan Tan
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  3. Junmin Peng
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Corresponding author

Correspondence to Junmin Peng .

Editor information

Editors and Affiliations

  1. Department of Psychiatry, University of Florida McKnight Brain Institute, Gainesville, Florida, USA

    Firas H. Kobeissy

  2. Department of Cell Biology, Microbiology, & Molecular Biology, University of South Florida, Tampa, Florida, USA

    Stanley M. Stevens, Jr.

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Bai, B., Tan, H., Peng, J. (2017). Quantitative Phosphoproteomic Analysis of Brain Tissues. In: Kobeissy, F., Stevens, Jr., S. (eds) Neuroproteomics. Methods in Molecular Biology, vol 1598. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-6952-4_8

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  • DOI: https://doi.org/10.1007/978-1-4939-6952-4_8

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-6950-0

  • Online ISBN: 978-1-4939-6952-4

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