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Realised by ALMS™
developer of the AIDS-HIV Reference project
Abstract No.: TuP-001
Session: Biomolecular Assemblies
Presentation date: Tue, Aug 29, 2006
Presentation time: 09:50 – 11:20

Characterization of Tetraspanin-enriched Membrane Microdomains by Mass Spectrometry: Composition, Post-translational Modifications and Organization

Magali Andre1, Julia Chamot-Rooke2, Claude Boucheix1, Eric Rubinstein1, Francois Le Naour1

1 Inserm U602, Villejuif, France
2 Ecole Polytechnique, DCMR, Palaiseau, France

Correspondence address: Magali Andre, Inserm U602, 14 avenue Paul Vaillant Couturier, Villejuif, 94800 France.

Keywords: Cross-linking; Glycosylation; Mass Spectrometry, High Resolution; Protein Identification.

Novel aspect: Identification of new components of the tetraspanin complexes, mass fingerprinting of complex peptide mixture from membrane proteins by FTICR-MS, in-situ cross-linking on living cells for structure determination, glycosylation and FTICR-MS.

 

Tetraspanins are integral membrane proteins involved in a variety of physiological and pathological processes. In cancer, clinical and experimental studies have reported a link between tetraspanin expression levels and metastasis. We have suggested that tetraspanins play a role as organizers of novel types of membrane microdomains including numerous other membrane proteins in a molecular network also called 'tetraspanin web'. Our aim was to address the composition and the organization of the complexes using proteomics.
To approach the composition, we have performed a proteomic characterization of the tetraspanin web in human colon cancer cells derived from primary tumor and metastasis from the same patients. The tetraspanin complexes were isolated after immunoaffinity purification using monoclonal antibodies directed against the tetraspanin CD9 and associated proteins were identified using LC-ESI-MS/MS and MALDI-FTICR-MS. The high resolution and mass accuracy of FTICR-MS allowed reliable identification using mass finger printing with only two peptides. Thus it could be used to resolve the composition of complex peptide mixtures from membrane proteins. This led to identify a large variety of membrane proteins such as adhesion molecules, enzymes and proteases, receptors and signalling molecules, membrane fusion proteins as well as poorly characterized proteins.1,2
Furthermore, we observed a variation of the molecular mass for some components between colon tumors and healthy tissue, suggesting post-translational modifications in cancer. We thus focused on glycosylation of CD9P-1, a major component of the tetraspanin web, which exhibits 25kDa of N-glycans out of a molecular mass of 125kDa. After N-glycosidase treatment, a mass finger printing using nano-ESI-FTICR has been performed. We looked for peptides exhibiting a conversion from Asn to Asp due to N-glycosydase digestion (1Da mass variation). Among the 9 potential sites for N-glycoyslation of CD9P-1, 8 were actually glycosylated with a partial occupancy for one of them. The glycans have been studied using lectin-blot and mass spectrometry.
We further addressed the organization of the complexes using in situ cross-linking experiments on living cells followed by cell lysis, immunoprecipitation, trypsin digestion and FTICR-MS. We had previously shown that each tetraspanin associates specifically with one or a few other membrane proteins forming primary complexes. We first focused on the primary complex constituted by the tetraspanin CD9 and its specific partner CD9P-1. This revealed the presence of CD9P-1 homodimers and CD9/CD9P-1 oligomers at the cell surface. This strategy allows characterization of the regions in close proximity. These studies revealed variations of the composition and post-translational modifications of the complexes during malignancy.

1. Andre et al., Proteomics in press (2006).
2. Le Naour et al., Mol. Cell. Proteomics in press (2006).