Improving MALDI-TOF-MS sample preparation for the analysis of affinity ligands selected from the screening of one bead-one peptide combinatorial libraries

MARÍA C. MARTÍNEZ CERON; SILVANA L. GIUDICESSI; MARIELA M. MARANI; FERNANDO ALBERICIO; OSVALDO CASCONE; ROSA ERRA-BALSELLS; SILVIA CAMPERI

Barcelona, Espana

Congreso; 14th European Congress on Biotechnoligy, Barcelona; 2009

Eoropean Society for Biotechnology

Improving MALDI-TOF-MS sample preparation for the analysis of affinity ligands selected from the screening of one bead-one peptide combinatorial libraries                                                                                                         María C. Martínez Ceron, † Silvana L. Giudicessi, ‡  Mariela M. Marani, † Fernando Albericio, *,#,∫ Osvaldo Cascone, † Rosa Erra-Balsells, ‡ Silvia A. Camperi. † †Cátedra de Microbiología Industrial y Biotecnología. Facultad de Farmacia y Bioquímica. Universidad de Buenos Aires. Junín 956, (1113) Ciudad Autónoma de Buenos Aires, Argentina. ‡CIHDECAR-CONICET. Departamento de Química Orgánica. Facultad de Ciencias Exactas y Naturales. Universidad de Buenos Aires. Pabellón II-Ciudad Universitaria, (1428) Ciudad Autónoma de Buenos Aires, Argentina. §Proteomics Platform, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain. #Institute for Research in Biomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028 Barcelona, Spain. ∫CIBER-BBN, Networking Centre on Bioengineering, Biomaterials and Nanomedicine, Barcelona Science Park, Baldiri Reixac 10, 08028-Barcelona, Spain. Affinity chromatography (AC) is the most effective method for the direct isolation and purification of biomolecules from complex mixtures. Successful separation by AC requires the availability of a specific ligand. Small peptides as AC 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". (each bead displays only one peptide entity). Peptide ligands can be selected from screening of the library against specific targets. Positive beads are isolated and the peptide 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. The success of MALDI measurements is partly empirical and depends on the type of matrix and proper sample preparation before MALDI analysis. Contaminants, matrix clusters and metal ion adducts interfere with peptide ionization and mass spectrum interpretation. In this work we analyzed different strategies to improve the analysis of positive beads by MALDI-TOF/TOF MS. A combinatorial library was synthesised on the HMBA-ChemMatrix resin by the DCR method using Fmoc chemistry. After library screening positive beads were isolated for its analysis. We replaced the guanidine usually used for bead washing before peptide analysis with a mixture of acetonitrile (MeCN), acetic acid (AcOH) and H2O (3:4:3). Removal of guanidine as contaminant improved matrix crystallization and peptide ionization. We optimized peptide-bead cleavage and peptide elution: 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 of AcOH- MeCN-H2O (3:4:3) and 0,5 µl of 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 matrixes were tested: a-cyano-4-hydroxycinnamic acid (CHCA) and 2,5-dihydroxybenzoic acid (DHB). The optimum concentration for CHCA was between 2-5 mg/ml and for DHB between 5-10 mg/mL. CHCA induced higher peptide ionization, but CHCA clusters sometimes interfere with MS spectrum interpretation. With DHB the matrix clusters were reduced but also the peptide ionization and the MSMS spectrum had very few peaks to deduced peptide sequence. The commercial ionic liquid matrices (ILM) CHC-1-butylamine and CHC-diethylamine were also used for comparative experiments.   To minimize clusters and adducts, serine or NH4H2PO4 were added to the sample matrix mixture at a concentration of 20 and 10 mM respectively and signal-to-noise ratio was increased.