This Platform continued its two activities, involving chemical protein microsequencing and protein analysis using ProteinChip technology (Bio-Rad). These activities are performed for the benefit of laboratories both inside and outside the Pasteur Institute.

Protein Microsequencing

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. Post-traductional 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 2010, we sequenced 435 samples for 48 laboratories (including 17 laboratories outside the Pasteur Institute and 79 samples).

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 2010 we performed analysis for 44 users.

Keywords: Proteins, Microsequencing, ProteinChip Arrays, SELDI, Mass Spectrometry

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.

T. Krey, J. D'Alayer, C.M. Kikuti, A. Saulnier, L. Damier-Piolle, I. Petitpas, D.X. Johansson, R.G. Tawar, B. Baron, B. Robert, P. England, M.A.A. Persson, A. Martin, F.A. Rey (2010). The disulfide bonds in glycoprotein e2 of hepatitis C virus reveal the tertiary organization of the molecule. PLoS Pathogens. vol 6, No 2, p e1000762

V. Monteil, A. Kolb, J. DAlayer, P. Biguin, and F. Norel (2010).Identification of Conserved Amino Acid Residues of Salmonella σ^S Chaperone Crl Involved in Crl-σ^S Interactions.Journal of Bacteriology, Feb. 2010, p. 1075-1087.

M. Beraud, A. Kolb, V. Monteil, J. DAlayer, and F. Norel (2010). A proteomic analysis reveals differential regulation Of the σS-dependent yciGFE(katN) locus by YncC and H-NS in Salmonella and Escherichia coli K-12.Mol Cell Proteomics.9 (12): 2601-2616.