MARANI mariela Mirta
congresos y reuniones científicas
A fast and inexpensive strategy to accelerate the affinity peptide ligands identification by direct MALDI-TOF-MS analysis of peptide combinatorial libraries
Barcelona, ESPAÑA
Congreso; 13th European Congress on Biotechnology; 2007
Institución organizadora:
European Federation of Biotechnology
Affinity chromatography (AC) it is the most effective method for the direct isolation and purification of biomolecules from complex mixtures. Short peptides are excellent ligands for AC as they are not likely to cause an immune response in case of leakage into the product, they are more stable than antibodies to elution and cleaning conditions and they usually have very acceptable selectivity (1). The combinatorial synthesis of peptide libraries allows obtaining millions of peptides empirically, thus greatly facilitating the discovery of suitable ligands for any given protein of interest. The divide–couple–recombine (DCR) method for library preparation assures an even representation of the library members and a one-bead-one-compound distribution (2). Although peptides are typically identified by Edman microsequencing, this method is time-consuming, expensive and only convenient for short- to medium-length peptides with a sensitivity of 1-5 pmol.  The aim of this work was to develop a fast and inexpensive strategy for the identification of peptide ligands by direct matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) analysis of peptide-beads screened from one-bead-one-peptide combinatorial libraries.  Streptavidin was used as the model protein. The linker 4-hydroxymethylbenzoic acid (HMBA) (3) was immobilised on ChemMatrixTM (4) resin.  Five residues of Gly were incorporated at the C-terminal to increase the final peptide molecular weight. A one-bead-one-peptide library of the nonapeptide XXXXGGGGG, where X = Ala, Phe, Gly, His, Leu, Pro, Gln, Arg, Val, was synthesised on the HMBA-ChemMatrixTM resin (3) by using the DCR method. The library had 6,561 different peptides (94). Positive control peptides with HPQ motif and negative control peptides without HPQ motif were also synthesised.  Screening was carried out with streptavidin-peroxidase conjugate. 4-Cl–naphtol and H2O2 was then added and the positive beads turned violet. Positive beads were isolated and washed with 8 M guanidine hydrochloride. The beads were sliced into two or four pieces, deposited onto the stainless-steel MALDI sample plate and treated with ammonia vapour to release the peptides. Cleaved peptides were eluted from the bead with acetic acid/acetonitrile/H2O. All the samples were analysed by MALDI-TOF-MS. 26 beads picked at random from the library were also analysed. Control peptides without HPQ motif did not show a positive reaction while control peptides with HPQ motif turned violet demonstrating that the linker and the five Gly residues had not impaired the specific binding nor facilitated unspecific binding. The 26 beads picked at random were analysed by MALDI-TOF-MS and peptides sequences were unambiguously identified with very good reproducibility between the bead pieces, thus evidencing the good homogeneity of the bead. Five positive beads were obtained from the library screening. Sequences HPQFGGGGG (two times), FHPQGGGGG (two times) and LHPQGGGGG were determined fully agreeing with Lam et al. (5) who described the high affinity of this sequence for streptavidin. This development will accelerate the identification of synthetic affinity peptide ligands for the purification of the new biopharmaceuticals introduced by the biotechnology industry. 1) P.Y. Huang, R.G. Carbonell, Biotechnol. Bioeng. 63 (1999) 633.  2) K.S. Lam, S.E. Salmon, E.M. Hersh, V.J. Hruby, W.M. Kazmierski, Nature. 354 (1991) 82. 3) S. Côte, PCT/cd 2004//001464, patent pending Int. 4) E. Atherton, C.J. Logan, R.C. Sheppard, Peptide synthesis. J. Chem. Soc. Perkin Trans. 1(1981) 538. 5) K.S. Lam, M. Lebl. ImmunoMethods. 1 (1992) 11.