Development of new approaches by mass spectrometry for the study of membrane proteins

Development of membrane proteomic tools

Membranes play a critical role in cellular structure by providing a physical barrier between the cell and its environment as well as between intracellular compartments. On the same way, membrane proteins (MPs) confer unique compartment specific functions and communication between separated environments. For example, plasma membrane proteins regulate the exchange of information through signaling mechanisms and the mediation of transport of ions and solutes. Because of the multitude and importance of biological functions they play, they also represent the largest class of protein targets for the pharmaceutical and biotechnological industries. Today, they account for approximately 60% of pharmaceutical drug targets since they are implicated in cancer, diabetes, epilepsy, neurodegenerative disorders or endocrinological dysfunctions. Our understanding of these diseases and thus our action-reaction possibilities are however hampered by the lack of information at the molecular level on the proteins involved. A detailed characterization of these receptors and of their native partners is a scientific challenge that would likely increase our understanding of human pathophysiology and lead to the discovery of novel pharmaceutical tools.

 To achieve this goal, we propose to use mass spectrometry (MS) and to take advantages of its unique abilities in identification of mutations or post-translational modification characterisation. Although proteomics technologies have made rapid progress in the analyses of soluble proteins in recent years, membrane proteins are largely under-represented in datasets, the major obstacles being related to their very amphiphilic nature and often lower abundance. Therefore, these issues cannot be addressed by extrapolating the existing high-throughput proteomic programs since they almost exclusively deal with soluble protein targets. MS signals are poor in number as well as in intensity, and the resulting sequence coverage very low.

Why is it so? Where are the undetected proteolytic peptides lost? During digestion? During gel extraction? During MS analysis? Is there a common feature between those peptides?

 As a result, no well-defined, widely-applicable protocols are available to date to achieve membrane subproteome and this is precisely what we would like to develop in the present project. 

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