|Protein Microsequencing and Analysis|
|HEAD||Dr. D’ALAYER Jacques / email@example.com|
|MEMBERS||Dr. EXPERT-BEZANCON Nicole
This Platform continued its two activities, involving chemical protein microsequencing and protein analysis using ProteinChip technology (Ciphergen / BioRad). These activities are performed for the benefit of laboratories both inside and outside the Pasteur Institute.
The chemical protein sequencing remains an important part of its work. Amino acid sequences are determined from purified or partially purified proteins or peptides. But we can also obtained internal sequences by cleaving proteins into fragments and purifying them by HPLC chromatography before sequencing. Postracductional modifications of amino acids can be also found by this technology (phosphorylation, methylation, acylation, glycosylation…).
Amino-acid sequences are performed in automatic sequencer using Edman chemical degradation (two ABI 494 from Applied Biosystems are available).
In 2007, we sequenced 428 samples for 46 laboratories (including 19 laboratories outside the Pasteur Institute).
The Platform also provides support services including advice about micro technics requisite to prepare samples before sequencing.
Protein Analysis (ProteinChip technology)
This technology combines the capture of proteins on different surfaces (ProteinChip arrays. An array consists of a metal base, with 8 active spots. It is shown below), and their analysis by mass spectrometry (SELDI-TOF-MS).
Each array contains chemical or biochemical surface (cationic, anionic, hydrophobic, hydrophilic or preactivated for covalent binding…).
These arrays are used to selectively bind whole classes of proteins even in crude sample. After appropriate washes, molecular weights of coated proteins are detected in the ProteinChip reader (SELDI-TOF-MS).
The possibilities of this technology are quite versatile. For instance: specific molecular interaction studies, biomarkers discovery, phosphorylation or glycosylation studies.
These analysis are carried out with very small amount of biological material, at a picomolar level.
In 2007 we performed analysis for 55 users.
Keywords: Proteins, Microsequencing, ProteinChips, Mass spectrometry
E. Real, J.C. Rain, V. Battaglia, C. Jallet, P. Perrin, N. Tordo, P. Chrisment, J. d'Alayer, P. Legrain, Y. Jacob.(2004) Antiviral Drug Discovery Strategy Using Combinatorial Libraries of Structurally Constrained Peptides. Journal of Virology, July, pp 7410 - 7417 PMID: 15220414
T. Freire, J. d'Alayer and S. Bay.(2006) Efficient Monitoring of Enzymatic Conjugation Reaction by Surface-Enhanced Laser Desorption/Ionisation Time of Flight Mass Spectrometry for Process Optimization. Bioconjugate Chem., vol 17, pp 559-564
E. Ferquet, L. de Haro, V. Jan, I. Guillemin, S. Jourdain, A. Teynié, J. d'Alayer, V. Choumet (2007) Reappraisal of Vipera aspis Venom Neurotoxicity. PLoS ONE. vol 2, No 11, pp e1194
J. d'Alayer, N. Expert-Bezançon and P. Beguin. (2007) Time-and temperature-dependent acetylation of the chemokine RANTES produced in recombinant Escherichia coli. Protein Expression and Purification, vol 55, No 1, pp 9-16
D. Fehr, S. E. Burr, M. Gilbert, J. d'Alayer, J. Frey, and M. Popoff (2007) Aeromonas Exoenzyme T of Aeromonas salmonicida is a Bifunctional Protein That Targets the Host Cytoskeleton The Journal of Biological Chemistry, vol 282, No 39, pp 28843-28852
Activity Reports 2007 - Institut Pasteur
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