MCF-7/BOS cells membrane proteome: comparison of two isolation methods using mass spectrometry

Membrane proteins (MP) play an important role in biological processes. Isolation and quantification of these MP using classical techniques is often limited due to their poor solubility and relatively low abundance. These membrane proteins enclosed markers which could be potential therapeutic targets. These potential therapeutic targets have to be accessible to antibodies and need to be presented in the plasmic membrane.
Assays were conducted on MCF-7 / BOS cell line, immortal and easier to cultivate. The goal of this work is to obtain an enriched membrane fraction to facilitate the analysis of the sample and to simplify the complex proteins mixture.
To isolate transmembrane proteins, we compared two methods. The first one used different extraction cycles characterized by different buffers to isolate membrane proteins. The second method labelled accessible extracellular domains at the surface of MCF-7 cells with biotin prior to differential centrifugation. The obtained enriched membrane proteome was digested with trypsin and/or Lysyl Endopeptidase. Obtained peptides were separated by 2D-HPLC chromatography and on-line analysed ion trap mass spectrometer, the Esquire HCT.