Protocol

Shotgun Proteomics

Volume 1156 of the series Methods in Molecular Biology pp 157-174

Date:

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

  • Andy ChristoforouAffiliated withDepartment of Genetics, University of Cambridge Email author 
  • , Alfonso Martinez AriasAffiliated withDepartment of Genetics, University of Cambridge
  • , Kathryn S. LilleyAffiliated withDepartment of Genetics, University of Cambridge

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Abstract

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