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Analysis of Brain Phosphoproteome Using Titanium Dioxide Enrichment and High-Resolution LC-MS/MS

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Current Proteomic Approaches Applied to Brain Function

Part of the book series: Neuromethods ((NM,volume 127))

Abstract

This chapter outlines the process of performing phosphoproteomic studies of brain tissue: brain dissection, protein extraction and digestion, phosphopeptide enrichment, and peptide identification and quantification by LC-MS/MS. We describe a refined method for rapid, simple, and efficient TiO2-based phosphopeptide enrichment that relies on specific binding of the peptidyl phosphate group and TiO2, with free phosphate competitor added to reduce nonspecific binding. Integration of such a robust phosphopeptide enrichment method, powerful high-resolution LC-MS/MS, and multiplex isobaric labeling enables deep profiling of phosphoproteome with high sensitivity from biological samples, such as the human brain.

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Acknowledgments

The authors thank Drs. Ping-Chung Chen and Yun Jiao for helpful discussion. This work was partially supported by National Institutes of Health grants R01GM114260, R01AG047928, R01AG053987, and ALSAC (American Lebanese Syrian Associated Charities). The MS analysis was performed in the St. Jude Children’s Research Hospital Proteomics Facility, partially supported by NIH Cancer Center Support Grant (P30CA021765).

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Authors and Affiliations

  1. Department of Structural Biology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Jeffrey M. Sifford, Hong Wang & Junmin Peng

  2. Department of Neurodevelopmental Biology, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Jeffrey M. Sifford, Hong Wang & Junmin Peng

  3. St. Jude Proteomics Facility, St. Jude Children’s Research Hospital, Memphis, TN, 38105, USA

    Haiyan Tan & Junmin Peng

Authors
  1. Jeffrey M. Sifford
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  2. Haiyan Tan
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  3. Hong Wang
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  4. Junmin Peng
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Corresponding author

Correspondence to Junmin Peng .

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Editors and Affiliations

  1. Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain

    Enrique Santamaría

  2. Clinical Neuroproteomics Unit, Navarrabiomed, Navarra Health Department, Public University of Navarra, Proteored-Institute of Health Carlos III (ISCIII), Navarra Institute for Health Research (IdiSNA), Pamplona, Spain

    Joaquín Fernández-Irigoyen

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Sifford, J.M., Tan, H., Wang, H., Peng, J. (2017). Analysis of Brain Phosphoproteome Using Titanium Dioxide Enrichment and High-Resolution LC-MS/MS. In: Santamaría, E., Fernández-Irigoyen, J. (eds) Current Proteomic Approaches Applied to Brain Function. Neuromethods, vol 127. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7119-0_9

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  • DOI: https://doi.org/10.1007/978-1-4939-7119-0_9

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-7118-3

  • Online ISBN: 978-1-4939-7119-0

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