CONICET | Buscador de Institutos y Recursos Humanos

Affinity chromatography (AC) is the most effective method for biomolecules purification from complex mixtures. Successful separation by AC requires availability of selective ligands. Small peptides as AC ligands are more selective than dyes and metals and more stable than antibodies. Divide-couple-recombine (DCR) method allows obtaining libraries with all possible combinations of the amino acids in the form of "one bead-one peptide". Peptide ligands can be selected from library screening against specific targets. Positive beads are isolated and peptides usually identified by Edman microsequencing. As this technique is expensive and time-consuming, we previously reported a rapid and inexpensive method based on MALDI-TOF MS analysis. Success of MALDI measurements is partly empirical and depends on the matrix type and proper sample preparation before MALDI analysis. Contaminants, matrix clusters and metal ion adducts interfere with peptide ionisation and mass spectrum interpretation. Herein we analyse different strategies to improve the positive beads analysis by MALDI-TOF/TOF MS. A combinatorial library was synthesised on the HMBA-ChemMatrix resin by the DCR method. After library screening, positive beads were isolated for their analysis. Guanidine, usually utilised for bead washing before peptide analysis, was replaced by a mixture of acetonitrile (MeCN), acetic acid (AcOH) and H2O (3:4:3). Removal of guanidine as contaminant improved matrix crystallisation and peptide ionisation. Peptide-bead cleavage and peptide elution were also optimised: beads were placed into microtubes in a drying chamber together with a flask containing NH4OH. Cleaved peptides were eluted from the beads with 20 µl AcOH- MeCN-H2O (3:4:3) and 0.5 µl sample was loaded onto the sample plate. Elution of peptides in microtubes instead of placing the bead in the sample plate increased sample aliquots in case spectrum had to be repeated. Two matrices were tested: alpha-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). Optimum concentrations for CHCA and DHB were 2-5 and 5-10 mg/ml respectively. CHCA induced higher peptide ionisation, but CHCA clusters sometimes interfere with MS spectrum interpretation. With DHB the matrix clusters were reduced but also the peptide ionisation and the MSMS spectrum had too few peaks to deduce the peptide sequence. The commercial ionic liquid matrices (ILM) CHC-1-butylamine and CHC-diethylamine were also used for comparison. By addition of serine to the sample matrix mixture at a concentration of 20 mM, formation of clusters and adducts was minimised and signal-to-noise ratio increased significantly.

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