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Large-Scale Protein Phosphorylation Analysis by Mass Spectrometry-Based Phosphoproteomics

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Protein Kinase Technologies

Part of the book series: Neuromethods ((NM,volume 68))

Abstract

Protein phosphorylation is one of the key mechanisms controlling cellular signaling networks. Due to the low abundance of phosphorylated proteins and weaker ionization efficiency of phosphopeptides during mass spectrometric analyses, it is highly required to remove abundant non-phosphopeptides from complex mixtures, such as cell lysates, allowing successful detection of low abundant phosphopeptides. We recently developed an aliphatic hydroxy acid-modified metal oxide chromatography (HAMMOC) to efficiently and selectively enrich phosphopeptides prior to mass spectrometry (MS) analysis. Here we describe a detailed workflow of HAMMOC for enriching phosphopeptides from small amounts, e.g., 100 μg, of tryptic digests of whole cell lysate. We also discuss the importance of confidently assigning phosphorylation site(s) from an identified phosphopeptide after MS analyses.

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Author information

Authors and Affiliations

  1. Graduate School of Pharmaceutical Sciences, Kyoto University, Kyoto, Japan

    Wei-Chi Ku & Yasushi Ishihama

  2. Institute for Advanced Biosciences, Keio University, Yamagata, Japan

    Naoyuki Sugiyama & Yasushi Ishihama

Authors
  1. Wei-Chi Ku
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  2. Naoyuki Sugiyama
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  3. Yasushi Ishihama
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Corresponding author

Correspondence to Yasushi Ishihama .

Editor information

Editors and Affiliations

  1. Biosignal Research Center, Kobe University, Rokkodai 1-1, Kobe, 657-8501, Japan

    Hideyuki Mukai

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Ku, WC., Sugiyama, N., Ishihama, Y. (2012). Large-Scale Protein Phosphorylation Analysis by Mass Spectrometry-Based Phosphoproteomics. In: Mukai, H. (eds) Protein Kinase Technologies. Neuromethods, vol 68. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-61779-824-5_3

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  • DOI: https://doi.org/10.1007/978-1-61779-824-5_3

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  • Publisher Name: Humana Press, Totowa, NJ

  • Print ISBN: 978-1-61779-823-8

  • Online ISBN: 978-1-61779-824-5

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