To screen one-bead-one-compound (OBOC) combinatorial libraries, tens of thousands to millions of compound beads are first mixed with a target molecule. The beads that interact with this molecule are then identified and isolated for compound structure determination. Here we describe an OBOC peptide library screening using streptavidin (SA) as probe protein, labeled with a red fluorescent dye and using the COPAS BIO-BEAD flow sorting equipment to separate fluorescent from nonfluorescent beads. The red dyes used were ATTO 590 and Texas Red. After incubating the library with the SA-red fluorescent dye conjugate, we isolated positive beads caused by peptide-SA interaction and false positive beads produced by peptide fluorescent dye interaction. These false positives were a drawback when sorting beads by COPAS. However,an in depth analysis of both kinds of beads allowed the differentiation of positives from false positives. The false positive beads showed bright homogeneous fluorescence, while positive beads had a heterogeneous fluorescence, exhibiting a characteristic halo appearance, with fluorescence intensity greatest at the bead surface and lowest in the core. The difference was more evident when using Texas Red instead of ATTO 590. Thus, positive beads could be manually separated from false positive ones. The beads were analyzed by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). Most of the sequences obtained from positive beads had the His-Pro-Gln motif. Peptides from false positive beads were rich in Leu/Ileu, His, Phe, and Tyr.

Antimicrobial peptides (AMPs), also known as host defense peptides, are small and mostly polycationic molecules that form part of the innate immune response. There are currently more than 3000 experimentally reported AMPs. Particularly in frogs, the temporin family has been discovered as potential AMPs. The aim of this work is to review the latest publications about this class of peptides, discuss their properties, and present an update of the last studies and new discoveries in the field. More than 130 temporins have been identified in this family. The most studied temporins are temporin A (TA), temporin B (TB), and temporin L (TL). These peptides showed antimicrobial activity against gram-negative, gram-positive bacteria and fungi. Since the discovery of temporins in 1996, several groups of researchers isolated different peptides from various species of frogs that were included as members of this family. Although antimicrobial activity of many temporins has not been analyzed yet, most of them showed antimicrobial and antifungal activities. A combination of nanotechnology and AMPs for temporins in different antimicrobial treatments could be a promising alternative for resistant pathogens. These studies demonstrate that, even with the advancement in scientific research on the composition and antimicrobial activity of temporins, further studies are necessary to wholly understand their components and mechanisms of action.

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Peptide KVPLITVSKAK was selected to design a synthetic ligand for affinity chromatography purification of recombinant human follicle stimulating hormone (rhFSH), based on the interaction of the hormone with the exoloop 3 of its receptor. The peptide was acetylated to improve its stability to degradation by exopeptidases. A cysteine was incorporated at the C‐termini to facilitate its immobilization to the chromatographic activated SulfoLink agarose resin. A sample of crude rhFSH was loaded to the affinity column, using 20 mM sodium phosphate, 0.5 mM methionine, and pH 5.6 and 7.2 as adsorption and elution buffers, respectively. The dynamic capacity of the matrix was 54.6 mg rhFSH/mL matrix and the purity 94%. The percentage of oxidized rhFSH was 3.4%, and that of the free subunits was 1.2%, both in the range established by the European Pharmacopeia, as also were the sialic acid content and the isoforms profile.

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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 HMBAChemMatrix 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 H 2 O (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 NH 4 OH. Cleaved peptides were eluted from the beads with 20 l AcOH-MeCN-H 2 O (3:4:3) and 0.5 l 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: ␣-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 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.