INVESTIGADORES
MARANI mariela Mirta
congresos y reuniones científicas
Título:
Optimization of MALDI-TOF MS methods for the analysis of peptide beads selected from the screening of one bead-one peptide combinatorial libraries
Autor/es:
MARTÍNEZ CERON MARÍA C.; GIUDICESSI SILVANA L.; MARANI MARIELA M.; CÔTÉ SIMON; ERRA-BALSELLS ROSA; ALBERICIO FERNANDO; CAMPERI SILVIA A.; CASCONE OSVALDO
Lugar:
Villa Carlos Paz, Córdoba, ARGENTINA
Reunión:
Congreso; SAIB XLIV Reunión anual (Sociedad Argentina de Investigación en Bioquímica y Biología Molecular); 2008
Institución organizadora:
SAIB
Resumen:
One bead-one peptide combinatorial libraries using divide-couple-recombine (DCR) method has become an important tool for new ligand identification. The method involves the synthesis of millions of peptides on beads so that each bead displays only one peptide entity. The library is screened against specific molecular targets. Positive beads are isolated and washed with guanidine hydrochloride for their analyses. We had reported a rapid and inexpensive strategy based on matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF) analysis of peptide beads. The peptide library was synthesized on the 4- hydroxymethylbenzoic acid (HMBA)-ChemMatrix resin. After screening, peptides were cleaved from the beads with ammonia vapor for MALDI analysis. Contaminants, matrix clusters and metal ion adducts interfere with peptide ionization and peptide mass spectrum interpretation. In this work we analyzed different strategies to improve MALDI spectra. We replaced the guanidine hydrochloride used for bead washing with a mixture of acetonitrile (ACN), acetic acid (AcOH) and H2O; elimination of guanidine as contaminant improved matrix crystallization.  We also optimized peptide-bead cleavage and peptide elution: beads were placed into microtubes in a desiccator together with a flask containing NH4OH. Cleaved peptides were eluted from the beads with 20 µl of AcOH- ACN-H2O and 0,5 µl of sample was loaded onto the sample plate.  The elution of the peptides in microtubes instead of placing the bead in the sample plate increased sample aliquots in case the spectrum had to be repeated. a-Cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB) matrix were tested. With CHCA higher peptide ionization was obtained. To minimize clusters, we examined washing and recrystallization of sample spot but that didn’t improve mass spectra. The addition of serine or ammonium monobasic phosphate to CHCA reduced matrix adducts what resulted in the increase of signal-to-noise ratio and an improvement in MALDI spectra.