Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by MALDI MS

M. C. MARTÍNEZ CERON; S. L. GIUDICESSI; M. M. MARANI; F. ALBERICIO; O. CASCONE; R. ERRA-BALSELLS; S. A. CAMPERI

ANALYTICAL BIOCHEMISTRY

Elsevier Science Ltd, The Netherlands

Año: 2009 vol. 400 p. 295 - 297

0003-2697

One-bead-one-peptide combinatorial libraries, an important tool for new ligand identification, involve the synthesis of millions of peptides on beads so that each bead displays only one peptide entity. After library screening against specific targets, positive beads are isolated and analysed by MALDI-TOF/TOF reflectron and MS/MS modes. Herein we studied different strategies to improve MALDI mass spectra. Guanidine, usually utilised for bead washing before peptide analysis, was replaced by MeCN/AcOH/H2O (3/4/3) (v/v/v), thus improving matrix crystallisation and peptide ionisation.  Peptide-bead bond cleavage and peptide elution were also optimised: beads were placed into microtubes in a drying chamber with a flask containing NH4OH or NH3/THF. NH4OH was as efficient as NH3/THF but cheaper and safer. Cleaved peptides were eluted from beads with 5-200 µL AcOH/MeCN/H2O (3/4/3) (v/v/v) and 1 µL sample was loaded onto the sample plate. Peptides elution in microtubes instead of placing beads in the sample plate yielded more sample aliquots when spectra had to be repeated.  For sample preparation, “successive dry layers deposit” showed to be better than “dried droplet method” (mixture method). Among the matrices analysed, a-cyano-4-hydroxycinnamic acid induced higher peptide ionisation. Formation of clusters was minimised by serine or NH4H2PO4 addition to the sample matrix mixture. EN EVALUACIÓN. FECHA DE ENVÍO 02/06/09.