INVESTIGADORES
CAMPERI Silvia Andrea
congresos y reuniones científicas
Título:
AFFINITY LIGAND SELECTION FROM THE SCREENING OF ONE-BEAD-ONE-PEPTIDE LIBRARIES FOLLOWED BY MASS SPECTROMETRY SEQUENCING
Autor/es:
S. A. CAMPERI; M. M. MARANI; N. B. IANNUCCI; S. CÔTÉ; F. ALBERICIO; O. CASCONE
Lugar:
Village Rio das Pedras, Brasil.
Reunión:
Congreso; Enpromer 2005; 2005
Resumen:
Small peptides as affinity chromatography ligands are more selective than dyes and metals and more stable than antibodies. Divide-couple-recombine (DCR) method allows obtaining a library with all possible combinations of the amino acids in the form of "one bead-one peptide". Peptide ligands can be selected from immunoaffinity screening of the library and peptide beads showing a positive reaction are usually identified by Edman microsequencing. As this technique is expensive and time-consuming, the aim of this study was to design a rapid and non-expensive method for peptide identification using mass spectrometry sequencing (MS/MS). 4- Hydroxymethylbenzoic acid (HMBA) linker was attached to Aminomethyl-ChemMatrixR resin, which is a PEG based solid support. The benzyl ester, which forms the HMBA linker with the first amino acid, is stable to piperidine used for removal of the Fmoc group, and to TFA, making the linkage applicable to Fmoc or Boc chemistry. The HMBA inertness to TFA allowed side-chain deprotection without releasing the peptide from the resin. Peptide cleavage can be performed with ammonia due to the ChemMatrixR stability to nucleophilic conditions. A combinatorial library containing the octapeptide XXXXGGGG where X=A, P or R was synthesised on the HMBA- ChemMatrixR resin by the DAR method using Fmoc chemistry. Side-chain deprotection was carried out with TFA. The immunoaffinity screening of the solid-supported library for an anti-Granulocyte-Macrophage-Colony-Stimulating Factor (GM-CSF) mAb was performed as usual. Beads showing a positive reaction to anti GM-CSF mAb were isolated. After cleavage of the peptides with ammonia/THF vapour, they were sequenced by electrospray-ionisation MS/MS, showing the sequence APARGGGG as previously obtained using the Edman technique. The immunoaffinity screening was greatly facilitated by the amphiphilicity of the ChemMatrixR beads and the use of MS/MS assay reduced the cost and time of the analysis.
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