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Analysis of PINK1 and CaMKII Substrates Using Mass Spectrometry-Based Proteomics

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Analysis of Post-Translational Modifications and Proteolysis in Neuroscience

Part of the book series: Neuromethods ((NM,volume 114))

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Abstract

Mass spectrometry is a powerful tool for protein phosphorylation analysis. Collision-induced dissociation (CID) is a widely applied fragmentation method. Complementary fragmentation techniques such as electron transfer dissociation (ETD) and higher-energy C-trap dissociation (HCD) enhance the accurate elucidation of phosphorylation sites. Here we present proteomic approaches used for identifying phosphorylation sites of in vitro-phosphorylated neuroligin-1 (NL-1) and for identifying PINK1 substrates from outer mitochondrial membrane proteins. Technical details on how to identify phosphorylation sites using CID, ETD, and HCD fragmentation are described, including sample preparation, in-gel and in-solution protein digestion, peptide separation, and data acquisition.

Supported by the National Institute of Neurological Disorders and Stroke Intramural Research Program

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Author information

Authors and Affiliations

  1. Protein/Peptide Sequencing Facility, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), 35 Convent Drive, Bethesda, MD, 20892, USA

    Yan Li

  2. Biochemistry Section, Surgical Neurology Branch, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, MD, USA

    Lesley A. Kane

  3. Receptor Biology Section, National Institute of Neurological Disorders and Stroke (NINDS), National Institutes of Health (NIH), Bethesda, MD, USA

    Michael A. Bemben & Katherine W. Roche

  4. Department of Biology, Johns Hopkins University, Baltimore, MD, USA

    Michael A. Bemben

Authors
  1. Yan Li
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  2. Lesley A. Kane
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  3. Michael A. Bemben
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  4. Katherine W. Roche
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Corresponding author

Correspondence to Yan Li .

Editor information

Editors and Affiliations

  1. Department of Biology, University of Wisconsin-Stout, Menomonie, Wisconsin, USA

    Jennifer Elizabeth Grant

  2. Department of Microbiology, Biochemistry and Molecular Genetics, Rutgers University—New Jersey Medical School Cancer Center Newark, Newark, New Jersey, USA

    Hong Li

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Li, Y., Kane, L.A., Bemben, M.A., Roche, K.W. (2015). Analysis of PINK1 and CaMKII Substrates Using Mass Spectrometry-Based Proteomics. In: Grant, J., Li, H. (eds) Analysis of Post-Translational Modifications and Proteolysis in Neuroscience. Neuromethods, vol 114. Humana Press, New York, NY. https://doi.org/10.1007/7657_2015_85

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  • DOI: https://doi.org/10.1007/7657_2015_85

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-3470-6

  • Online ISBN: 978-1-4939-3472-0

  • eBook Packages: Springer Protocols

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