Determining Protein Subcellular Localization in Mammalian Cell Culture with Biochemical Fractionation and iTRAQ 8-Plex Quantification

  • Andy Christoforou
  • Alfonso Martinez Arias
  • Kathryn S. Lilley
Part of the Methods in Molecular Biology book series (MIMB, volume 1156)


Protein subcellular localization is a fundamental feature of posttranslational functional regulation. Traditional microscopy based approaches to study protein localization are typically of limited throughput, and dependent on the availability of antibodies with high specificity and sensitivity, or fluorescent fusion proteins. In this chapter we describe how Localization of Organelle Proteins by Isotope Tagging (LOPIT), a mass spectrometry based workflow coupling biochemical fractionation and iTRAQ™ 8-plex quantification, can be applied for the high-throughput characterization of protein localization in a mammalian cell culture line.

Key words

Isobaric tagging iTRAQ LOPIT Organelle proteomics Protein localization Spatial proteomics Subcellular localization 



The authors would like to thank Daniel Nightingale and Julie Howard for their helpful comments to improve the clarity of the protocol. A.C. was funded by BBSRC grant BB/D526088/1.


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Copyright information

© Springer Science+Business Media New York 2014

Authors and Affiliations

  • Andy Christoforou
    • 1
  • Alfonso Martinez Arias
    • 1
  • Kathryn S. Lilley
    • 1
  1. 1.Department of GeneticsUniversity of CambridgeCambridgeUK

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