Dr Manfredo Quadroni, MER

Activité de recherche :

• Marquage métabolique et spectrométrie de masse spécifique : outils pour étudier les changements dans l'expression des protéines

Mots clé :

• Protéomique, Spectrométrie de masse, synthèse des protéines

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Proteomics techniques based on mass spectrometry (MS) have become powerful tools to analyse cellular protein expression patterns. However, changes in protein expression following a biological stimulus often affect only a small set of proteins, can be limited in time and display strictly regulated kinetics. Such small-scale, temporary events often become undetectable against the background of the total proteome, unless special strategies are employed to highlight them.
We propose the development of a technique to specifically detect and identify newly synthesized proteins against a background of pre-existing ones. The approach will be based on time-limited metabolic labelling of cultured cells following a variation of the SILAC [1] scheme. Only proteins synthesized in the chosen time frame will have incorporated an isotope-labelled essential amino acid. After separation and digestion, peptides from such newly synthesized proteins are specifically detected and identified by a label-specific parent ion scan - MS/MS analysis on a QTRAP instrument.
Development of such a technique will necessitate the implementation of advanced labelling and mass spectrometry techniques as well as specific data analysis tools. One first application will be to analyse the infection of HeLa cells by Human Herpes simplex Virus 1 (HHSV-1). In this system, distinct and well-characterised sets of viral proteins are synthesised at different time points after infection, while synthesis of cellular endogenous proteins is strongly repressed. Other applications will address the study of secreted proteins.

References :

• Ong SE, Blagoev B, Kratchmarova I, Kristensen DB, Steen H, Pandey A, Mann M. Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Mol Cell Proteomics.

• Palagi PM, Walther D, Quadroni M, Catherinet S, Burgess J, Zimmermann-Ivol CG, Sanchez JC, Binz PA, Hochstrasser DF, Appel RD. MSight: an image analysis software for liquid chromatography-mass spectrometry. Proteomics. 2005 Jun;5(9):2381-4.

• Owen HR, Quadroni M, Bienvenut W, Buerki C, Hottiger MO., Identification of novel and cell type enriched cofactors of the transcription activation domain of RelA (p65 NF-kappaB). J Proteome Res. 2005 Jul-Aug;4(4):1381-90.