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Quantitation of Met Tyrosine Phosphorylation Using MRM-MS

  • Zhaojing Meng
  • Apurva K. Srivastava
  • Ming Zhou
  • Timothy Veenstra
Protocol
Part of the Methods in Molecular Biology book series (MIMB, volume 1002)

Abstract

Phosphorylation has long been accepted as a key cellular regulator of cell signaling pathways. The recent development of multiple-reaction monitoring mass spectrometry (MRM-MS) provides a useful tool for measuring the absolute quantity of phosphorylation occupancy at pivotal sites within signaling proteins, even when the phosphorylation sites are in close proximity. Here, we described a targeted quantitation approach to measure the absolute phosphorylation occupancy at Y1234 and Y1235 of Met. The approach is utilized to obtain absolute occupancy of the two phosphorylation sites in the full-length recombinant Met. It is further applied to quantitate the phosphorylation state of these two sites in SNU-5 cells treated with a Met inhibitor.

Key words

Met Phosphorylation Multiple-reaction monitoring Mass spectrometry Quantitation 

Notes

Acknowledgments

This project has been funded in whole or in part with federal funds from the National Cancer Institute, National Institutes of Health, under Contract HHSN261200800001E. The content of this publication does not necessarily reflect the views or policies of the Department of Health and Human Services, nor does mention of trade names, commercial products, or organizations imply endorsement by the United States Government.

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Copyright information

© Springer Science+Business Media, LLC 2013

Authors and Affiliations

  • Zhaojing Meng
    • 1
  • Apurva K. Srivastava
    • 1
  • Ming Zhou
    • 2
  • Timothy Veenstra
    • 2
  1. 1.Laboratory of Proteomics and Analytical Technologies, Advanced Technology Program, Frederick National Laboratory for Cancer Research, SAIC-Frederick, Inc.FrederickUSA
  2. 2.Laboratory of Proteomics and Analytical Technologies, SAIC-Frederick, Inc., Frederick National Laboratory for Cancer ResearchFrederickUSA

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