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