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ATM Kinase pp 197-213 | Cite as

Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling

  • Jeffrey R. Whiteaker
  • Lei Zhao
  • Regine M. Schoenherr
  • Jacob J. Kennedy
  • Richard G. Ivey
  • Amanda G. PaulovichEmail author
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1599)

Abstract

Peptide immunoaffinity enrichment coupled with targeted mass spectrometry is a quantitative approach for the robust and reproducible quantification of peptide analytes. The approach is capable of multiplexed quantification of peptides, including posttranslational modifications such as phosphorylation. Anti-peptide antibodies are used to enrich analytes and heavy stable isotope-labeled standards. The enriched peptides are directly measured by multiple reaction monitoring (MRM), a well-characterized quantitative mass spectrometry-based method. Quantification is performed by measuring the analyte (light) peptide response relative to the heavy standard, which is spiked at a known concentration. Here, we describe the methodology for multiplexed measurement of phosphorylated peptides on the ATM kinase and their nonmodified peptide analogs in cellular lysates. The method provides quantitative measurements of phospho-signaling and can be extended to a number of other phosphopeptides and sample types.

Key words

Targeted proteomics DNA damage response Multiplex Quantitation LC/MS 

Notes

Acknowledgments

This work was funded by the Clinical Proteomic Tumor Analysis Consortium (CPTAC) of the US National Cancer Institute (U24CA160034).

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Copyright information

© Springer Science+Business Media LLC 2017

Authors and Affiliations

  • Jeffrey R. Whiteaker
    • 1
  • Lei Zhao
    • 1
  • Regine M. Schoenherr
    • 1
  • Jacob J. Kennedy
    • 1
  • Richard G. Ivey
    • 1
  • Amanda G. Paulovich
    • 1
    Email author
  1. 1.Fred Hutchinson Cancer Research CenterSeattleUSA

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