17th International Mass Spectrometry Conference :: Prague, 2006
> Go to contents (site navigation)
|Presentation date:||Mon, Aug 28, 2006|
|Presentation time:||09:50 – 11:20|
Isabel D. Alves1, Diane Delaroche1, Emmanuelle S. Sachon1,2, Gerard Bolbach1,2, Solange Lavielle1, Sandrine Sagan11 Universite Pierre et Marie Curie-Paris6, UMR 7613, Paris F-75005, France
Correspondence address: Isabel D. Alves, Universite Pierre et Marie Curie, UPMC, CNRS-UMR 7613, case courier 182, 4 place Jussieu, Paris, 75232 cedex 05 France.
Keywords: Detergent; MALDI; Protein, Membrane-bound; Purification.
Novel aspect: Mass spectrometry analysis of intact membrane proteins.
Membrane proteins constitute an important class of proteins since they encode for about 30% of the human genome. Nonetheless the analysis by mass spectrometry of membrane proteins in their entire form is still challenging due in part to the difficulty in obtaining and purifying them in large quantities under conditions (buffer, salt, detergent) that are compatible with this technique and also derived from the dual nature of those proteins (both hydrophobic and hydrophilic).1 Most of the proteomic studies have been focused on the peptide fingerprint or sequence. Nonetheless, the analysis by mass spectrometry of the entire protein is advantageous because it allows for more precise information both in terms of the homogeneity and purity of the entire protein and also in the study of oxidation and post-translational modifications (phosphorylation, glycosylation, palmitoylation, etc.).
The neurokinin 1 (NK-1) receptor is a member of the G-protein coupled receptors (GPCRs). This receptor possesses an unusual pharmacological profile with the existence of two non-stoichiometric binding sites, the origin of which is so far unknown.2 In order to purify and detect this receptor a His-tag has been introduced in the C-terminal side and the receptor stably transfected into CHO cells with an expression level of 10 pmole/mg protein. The receptor has been extracted from the membrane by detergent solubilization and purified using magnetic beads functionalized with Co2+. The purified receptor has been analyzed by SDS page and western blot which show that the receptor is ~80% pure. Moreover the receptor was photolabeled with a radioactive SP analogue confirming the identity of the receptor. To better access the purity of the sample and the exact mass of the protein the analysis by mass spectrometry of the solubilized and purified receptor has been performed. The magnetic beads functionalized with Co2+ (containing the solubilized receptor) were spotted directly on the MALDI plate in the presence of HCCA and analyzed. The results point to the existence of two forms of glycosylated receptor with masses between 57,000-58,000 (mass of the unmodified receptor is about 49,000) and also the existence of a non-glycosylated form with a mass of 48,000. The treatment of these species with N-glycosidase F lead to a band shift to a lower mass as visualized by SDS page and western blot analysis confirming that the species correspond to the glycosylated receptor. This purification of the receptor by magnetic beads and its direct analysis on MALDI constitutes a rapid and efficient method for the analysis of membrane proteins. Studies to determine the origin of the two distinct receptor species are underway.
1. T. Rabilloud, Nat. Biotechnol. 21, 508 (2003).
2. I. D. Alves, D. Delaroche, B. Mouillac, Z. Salamon, G. Tollin, V. J. Hruby, S. Lavielle and S. Sagan, Biochemistry in press (2006).