INVESTIGADORES
MARANI Mariela Mirta
congresos y reuniones científicas
Título:
Improving MALDI-TOF-MS sample preparation for the analysis of affinity ligands selected from one-bead-one peptide combinatorial libraries screening
Autor/es:
MARTÍNEZ CERON MARÍA C.; GIUDICESSI SILVANA L.; MARANI MARIELA M.; ALBERICIO FERNANDO; CASCONE OSVALDO; ERRA-BALSELLS ROSA; CAMPERI SILVIA A.
Lugar:
Barcelona, España
Reunión:
Congreso; ECB 14th European Congress on Biotechnology; 2009
Resumen:
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 AcOHMeCNH2O
(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: α-cyano-4-hydroxycinnamic acid (CHCA) and
2,5-dihydroxybenzoic acid (DHB). Optimum concentrations for CHCA and
DHB were 25 and 510 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 the 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.