Protein Microsequencing and Analysis


  HEADDr D’ALAYER Jacques / jdalayer@pasteur.fr
  MEMBERSDr EXPERT-BEZANCON Nicole


  Annual Report

This Platform has two major activities: chemical protein microsequencing and protein analysis using ProteinChip technology (Ciphergen / BioRad). These activities are carried out as a service for laboratories both inside and outside the Pasteur Institute.

Protein Microsequencing

Chemical protein sequencing remains an important part of this Platform’s work. Amino-acid sequences are determined from purified or partially purified proteins or peptides. In addition, internal constituent amino acid sequences of proteins can be determined following protein cleavage and purification by HPLC chromatography prior to sequencing. Post-translational modification of amino acids can also be identified with this technology e.g. phosphorylation, methylation, acylation, glycosylation etc.

Amino-acid sequencing is done with automatic sequencers (Applied Biosystems ABI 494 and ABI 473A) using classical Edman chemical degradation.

In 2006, we processed 406 samples for 42 different laboratories, 16 of which were external.

The Platform also provides support services, including advice about micro-techniques for sample preparation before sequencing.

Ciphergen Protein-Chip analysis

This technology combines protein capture on different Protein-Chip surfaces followed by mass spectrometry analysis (SELDI-TOF-MS). Chip arrays consist of a metal base, with space for 8 samples, shown below. Arrays can have a variety of chemical or biochemical surfaces with different binding characteristics e.g. cationic, anionic, hydrophobic, hydrophilic, metal-chelated, or pre-activated for covalent binding etc.

They can be used to selectively bind whole classes of proteins, even in crude samples. After appropriate washing, molecular weights of bound proteins are detected in the ProteinChip reader (SELDI-TOF-MS).

This technology is particularly interesting because it has multiple quite diverse applications, including the analysis of many types of specific molecular interaction, biomarker discovery, post-translational modifications such as phosphorylation, acylation or glycosylation, and proteolytic processing, to name only a few.

Importantly, these analyses can be carried out with very small amounts of biological material, at nano or pico-molar levels.

In 2006 we analyzed samples for 38 different projects using the Ciphergen technology, and in 2007 we plan to undertake a campaign to better inform the Pasteur scientific community regarding the polyvalent possibilities of this technology.

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  Publications

Publications 2006 of the unit on Pasteur's references database




Activity Reports 2006 - Institut Pasteur
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