Recent developments in less known and multi-resistant fungal opportunists.

Fungal infections have increased in recent years due to host factors, such as oncohaematological and transplant-related disorders, immunosuppressive therapy, and AIDS. Additionally, molecular and proteomic facilities have become available to identify previously unrecognizable opportunists. For these reasons, reports on less-known and recalcitrant mycoses, such as those caused by black fungi, hyaline filamentous fungi, coelomycetes, Mucorales, and non-Candida yeasts have emerged. In this review, novel taxonomy in these groups, which often are multi-resistant to one or several classes of antifungals, is discussed. Clinical presentations, diagnosis and current treatment of some major groups are summarised.

A short peptide fragment of the vascular endothelial growth factor as a novel ligand for bevacizumab purification.

Bevacizumab is a vascular endothelial growth factor (VEGF)-directed monoclonal antibody (mAb) used for the treatment of several human cancers. Given that bevacizumab is administered intravenously, it must have extremely high purity, which is achieved by purification with protein A affinity chromatography (AC). However, protein A is a very expensive ligand, thereby increasing the cost of purification. Furthermore, the harsh elution conditions required to recover bevacizumab from the AC column can damage both the mAb and protein A. In contrast, short peptides show higher stability, easier synthesis and lower cost and are therefore ideal ligands for AC. In the present study, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment that binds bevacizumab, was synthesized and immobilized on agarose. The peptidyl-agarose showed affinity for bevacizumab, with an equilibrium dissociation constant value of 2.2±0.5 x 10-7 M under optimal conditions. Samples of CHO cell filtrate producing bevacizumab were loaded on the peptidyl-agarose chromatography column. Bevacizumab was recovered from the elution fraction with a yield of 94% and a purity of 98%. The maximum capacity (qm) 38±2 mg of bevacizumab per mL of matrix was comparable to that of commercial protein A matrices. Moreover, the peptide ligand showed greater stability and a lower cost than protein A. Unlike peptides previously reported for IgG purification, the ligand described herein allows mAb elution under mild conditions, thereby favoring the integrity of bevacizumab. The lack of Trp, Met or Cys in the peptide prevents its oxidation and extends the useful life of the chromatographic matrix.

An inhibitory mechanism of action of coiled-coil peptides against type three secretion system from enteropathogenic Escherichia coli.

Human pathogenic gram-negative bacteria, such as enteropathogenic Escherichia coli (EPEC), rely on type III secretion systems (T3SS) to translocate virulence factors directly into host cells. The coiled-coil domains present in the structural proteins of T3SS are conformed by amphipathic alpha-helical structures that play an important role in the protein-protein interaction and are essential for the assembly of the translocation complex. To investigate the inhibitory capacity of these domains on the T3SS of EPEC, we synthesized peptides between 7 and 34 amino acids based on the coiled-coil domains of proteins that make up this secretion system. This analysis was performed through in vitro hemolysis assays by assessing the reduction of T3SS-dependent red blood cell lysis in the presence of the synthesized peptides. After confirming its inhibitory capacity, we performed molecular modeling assays using combined techniques, docking-molecular dynamic simulations, and quantum-mechanic calculations of the various peptide-protein complexes, to improve the affinity of the peptides to the target proteins selected from T3SS. These techniques allowed us to demonstrate that the peptides with greater inhibitory activity, directed against the coiled-coil domain of the C-terminal region of EspA, present favorable hydrophobic and hydrogen bond molecular interactions. Particularly, the hydrogen bond component is responsible for the stabilization of the peptide-protein complex. This study demonstrates that compounds targeting T3SS from pathogenic bacteria can indeed inhibit bacterial infection by presenting a higher specificity than broad-spectrum antibiotics. In turn, these peptides could be taken as initial structures to design and synthesize new compounds that mimic their inhibitory pharmacophoric pattern.

Recombinant human follicle stimulating hormone purification by a short peptide affinity chromatography.

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.

Simple method to assess stability of immobilized peptide ligands against proteases.

Although peptides are used as affinity chromatography ligands, they could be digested by proteases. Usually, peptide stability is evaluated in solution, which differs from the resin-bounded peptide behavior. Furthermore, the study of the degradation products requires purification steps before analysis. Here, we describe an easy method to assess immobilized peptide stability. Sample peptides were synthesized on hydroxymethylbenzamide-ChemMatrix resin. Peptidyl-resin beads were then incubated with solutions containing proteases. Peptides were detached from the solid support with ammonia vapor and analyzed by matrix-assisted laser desorption/ionization and electrospray ionization mass spectrometry, allowing the detection of the whole peptides as well as their C-terminal degradation products. The method allowed a fast evaluation of peptide ligand stability in solid phase towards proteases that may be present in the crude sample before their use as ligands in affinity chromatography. Copyright © 2017 European Peptide Society and John Wiley & Sons, Ltd.

A simple protocol for combinatorial cyclic depsipeptide libraries sequencing by matrix-assisted laser desorption/ionisation mass spectrometry.

Short cyclic peptides have a great interest in therapeutic, diagnostic and affinity chromatography applications. The screening of 'one-bead-one-peptide' combinatorial libraries combined with mass spectrometry (MS) is an excellent tool to find peptides with affinity for any target protein. The fragmentation patterns of cyclic peptides are quite more complex than those of their linear counterparts, and the elucidation of the resulting tandem mass spectra is rather more difficult. Here, we propose a simple protocol for combinatorial cyclic libraries synthesis and ring opening before MS analysis. In this strategy, 4-hydroxymethylbenzoic acid, which forms a benzyl ester with the first amino acid, was used as the linker. A glycolamidic ester group was incorporated after the combinatorial positions by adding glycolic acid. The library synthesis protocol consisted in the following: (i) incorporation of Fmoc-Asp[2-phenylisopropyl (OPp)]-OH to Ala-Gly-oxymethylbenzamide-ChemMatrix, (ii) synthesis of the combinatorial library, (iii) assembly of a glycolic acid, (iv) couple of an Ala residue in the N-terminal, (v) removal of OPp, (vi) peptide cyclisation through side chain Asp and N-Ala amino terminus and (vii) removal of side chain protecting groups. In order to simultaneously open the ring and release each peptide, benzyl and glycolamidic esters were cleaved with ammonia. Peptide sequences could be deduced from the tandem mass spectra of each single bead evaluated. The strategy herein proposed is suitable for the preparation of one-bead-one-cyclic depsipeptide libraries that can be easily open for its sequencing by matrix-assisted laser desorption/ionisation MS. It employs techniques and reagents frequently used in a broad range of laboratories without special expertise in organic synthesis.

Fully Automated Screening of a Combinatorial Library to Avoid False Positives: Application to Tetanus Toxoid Ligand Identification.

Peptide ligands are widely used in protein purification by affinity chromatography. Here, we applied a fully automated two-stage library screening method that avoids false positive peptidyl-bead selection and applied it to tetanus toxoid purification. The first library screening was performed using only sulforhodamine (a fluorescent dye), and fluorescent beads were isolated automatically by flow cytometry and discarded. A second screening was then performed with the rest of the library, using the target protein (tetanus toxoid)-rhodamine conjugate. This time, fluorescent beads were isolated, and peptide sequences were identified by matrix-assisted laser desorption/ionization tandem mass spectrometry. Those appearing with greater frequency were synthesized and immobilized on agarose to evaluate a range of chromatographic purification conditions. The affinity matrix PTx1-agarose (Ac-Leu-Arg-Val-Tyr-His-Gly-Gly-Ala-Gly-Lys-agarose) showed the best performance when 20 mM sodium phosphate, 0.05% Tween 20, pH 5.9 as adsorption buffer and 100 mM Tris-HCl, 100 mM NaCl, pH 8.0 as elution buffer were used. A pure tetanus toxoid (Ttx) was loaded on a chromatographic column filled with the PTx1 matrix, and 96% adsorption was achieved, with a K d of 9.18 ± 0.07 nmol/L and a q m of 1.31 ± 0.029 µmol Ttx/mL matrix. Next, a Clostridium tetani culture supernatant treated with formaldehyde (to obtain the toxoid) was applied as a sample. The sodium dodecyl sulfate polyacrylamide gel electrophoresis analysis showed a band, identified by electrospray ionization mass spectrometry as the Ttx, that appeared only in the elution fraction, where an S-layer protein was also detected.

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification.

The interest in therapeutic monoclonal antibodies (mAbs) has significantly grown in the pharmaceutical industry, exceeding 100 FDA mAbs approved. Although the upstream processing of their industrial production has been significantly improved in the last years, the downstream processing still depends on immobilized protein A affinity chromatography. The high cost, low capacity and short half-life of immobilized protein A chromatography matrices, encouraged the design of alternative short-peptide ligands for mAb purification. Most of these peptides have been obtained by screening combinatorial peptide libraries. These low-cost ligands can be easily produced by solid-phase peptide synthesis and can be immobilized on chromatographic supports, thus obtaining matrices with high capacity and selectivity. Furthermore, matrices with immobilized peptide ligands have longer half-life than those with protein A due to the higher stability of the peptides. In this review the design and synthesis of peptide ligands, their immobilization on chromatographic supports and the evaluation of the affinity supports for their application in mAb purification is described.

Diamond, titanium dioxide, titanium silicon oxide, and barium strontium titanium oxide nanoparticles as matrixes for direct matrix-assisted laser desorption/ionization mass spectrometry analysis of carbohydrates in plant tissues.

Nanoparticles (NPs) of diamond, titanium dioxide, titanium silicon oxide, barium strontium titanium oxide, and silver (Ag) were examined for their potential as MALDI matrixes for direct laser desorption/ionization of carbohydrates, especially fructans, from plant tissue. Two sample preparation methods including solvent-assisted and solvent-free (dry) NPs deposition were performed and compared. All examined NPs except for Ag could desorb/ionize standard sucrose and fructans in positive and in negative ion mode. Ag NPs yielded good signals only for nonsalt-doped samples that were measured in the negative ion mode. In the case of in vivo studies, except for Ag, all NPs studied could desorb/ionize carbohydrates from tissue in both the positive and negative ion modes. Furthermore, compared to the results obtained with soluble sugars extracted from plant tissues, fructans with higher molecular weight intact molecular ions could be detected when the plant tissues were directly profiled. The limit of detection (LOD) of fructans and the ratios between signal intensities and fructan concentrations were analyzed. NPs had similar LODs for standard fructan triose (1-kestose) in the positive ion mode and better LODs in the negative ion mode when compared with the common crystalline organic MALDI matrixes used for carbohydrates (2,5-dihydroxybenzoic acid and nor-harmane) or carbon nanotubes. Solvent-free NP deposition on tissues partially improves the signal acquisition. Although lower signal-to-noise ratio sugar signals were acquired from the tissues when compared to the solvent-assisted method, the reproducibility averaged over all sample was more uniform.

Sample preparation for sequencing hits from one-bead-one-peptide combinatorial libraries by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Optimization of bead analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) after the screening of one-bead-one-peptide combinatorial libraries was achieved, involving the fine-tuning of the whole process. Guanidine was replaced by acetonitrile (MeCN)/acetic acid (AcOH)/water (H(2)O), improving matrix crystallization. Peptide-bead cleavage with NH(4)OH was cheaper and safer than, yet as efficient as, NH(3)/tetrahydrofuran (THF). Peptide elution in microtubes instead of placing the beads in the sample plate yielded more sample aliquots. Successive dry layers deposit sample preparation was better than the dried droplet method. Among the matrices analyzed, alpha-cyano-4-hydroxycinnamic acid resulted in the best peptide ion yield. Cluster formation was minimized by the addition of additives to the matrix.

Use of a phosphopeptide as a ligand to purify phospholipase A2 from the venom of Crotalus durisuss terrificus by affinity chromatography.

The venom of Crotalus durissus terrificus (Cdt) is a source of a wide variety of toxins, some of them with interesting pharmacological applications. Of these toxins, the phospholipase A2 (PLA2) subunit of crotoxin (Ctx) has been studied for its potential as an antiviral and antibacterial agent. Peptides have proven useful ligands for the purification of numerous molecules, including antibodies, toxins, enzymes and other proteins. Here, we sought to use a phosphopeptide (P-Lys) as a ligand for PLA2 purification. P-Lys was synthesized in solid phase on Rink-Amide-ChemMatrix resin, immobilized on NHS-agarose, and then evaluated as a chromatographic matrix. Under the best conditions, total protein adsorption reached 39% and only the eluate fraction presented PLA2 activity. Analysis of the eluate by SDS-PAGE showed three bands, one corresponding to the molecular weight of PLA2 (14 kDa). Said bands were analyzed by mass spectrometry and identified as PLA2 and its multimers. The final product showed a purity of over 90%. In addition, slightly changing the process conditions also allowed the isolation of crotamine.

Synthetic peptides to produce antivenoms against the Cys-rich toxins of arachnids.

Scorpion and spider envenomation is treated with the appropriate antivenoms, prepared as described by Césaire Auguste Phisalix and Albert Calmette in 1894. Such treatment requires the acquisition and manipulation of arachnid venoms, both very complicated procedures. Most of the toxins in the venoms of spiders and scorpions are extremely stable cysteine-rich peptide neurotoxins. Many strategies have been developed to obtain synthetic immunogens to facilitate the production of antivenoms against these toxins. For example, whole peptide toxins can be synthesized by solid-phase peptide synthesis (SPPS). Also, epitopes of the toxins can be identified and after the chemical synthesis of these peptide epitopes by SPPS, they can be coupled to protein carriers to develop efficient immunogens. Moreover, multiple antigenic peptides with a polylysine core can be designed and synthesized. This review focuses on the strategies developed to obtain synthetic immunogens for the production of antivenoms against the toxic Cys-rich peptides of scorpions and spiders.

Protocol for bevacizumab purification using Ac-PHQGQHIGVSK-agarose.

Bevacizumab is a monoclonal antibody, produced in CHO cells, used for the treatment of many human cancers. It is an anti-vascular endothelial growth factor (antsi-VEGF) that blocks the growth of tumor blood vessels. Nowadays its purification is achieved by affinity chromatography (AC) using protein A which is a very expensive ligand. On the other hand, the peptide Ac-PHQGQHIGVSK contained in the VEGF fragment binds bevacizumab with high affinity. This short peptide ligand has higher stability and lower cost than protein A and it can be prepared very easily by solid phase peptide synthesis. The present protocol describes the synthesis of Ac-PHQGQHIGVSK-agarose and its use for affinity chromatography purification of bevacizumab from a clarified CHO cell culture. •Ac-PHQGQHIGVSK-agarose capacity and selectivity are equivalent to those of protein A matrices.•The peptide ligand shows a greater stability and lower cost. The lack of Trp, Met or Cys in the peptide ligand prevents its oxidation and extends the useful life of the chromatographic matrix.•Mild conditions used during chromatography preserved the integrity of bevacizumab.

Screening of one-bead-one-peptide combinatorial library using red fluorescent dyes. Presence of positive and false positive beads.

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.

Single step recombinant human growth hormone (rhGH) purification from milk by peptide affinity chromatography.

Recombinant human growth hormone (rhGH) is used for the treatment of several pathologies, most of them related to growth. Although different expression systems can be used for its production, the milk from transgenic cows is one of the most interesting due to the high rhGH level achieved (5 g/L). We have designed and synthesized short peptides (9 or 10 amino acid long) using Fmoc chemistry and studied their ability to purify rhGH from milk once immobilized on an agarose support. Using spiked milk with the hormone as a sample, rhGH was purified with 88% yield and 92% purity in a single step with a fold purification of 4.5. © 2018 American Institute of Chemical Engineers Biotechnol. Prog., 34:999-1005, 2018.

Latest Advances in OBOC Peptide Libraries. Improvements in Screening Strategies and Enlarging the Family From Linear to Cyclic Libraries.

Solid phase screenings of one bead one compound (OBOC) libraries have been widely used to find ligands with pharmacological and analytical uses, and to purify or detect proteins in complex mixtures. To improve library screening, in the last years various strategies have been developed to avoid the selection of false positive beads and to obtain selective ligands. Currently, there is great interest in cyclic peptides because of their resistance to enzymatic degradation and higher selectivity compared to their linear counterparts. Lots of cyclic peptide libraries protocols have been recently developed to facilitate hits analysis. The aim of this review is to summarize the latest applications of solid phase screening of OBOC combinatorial peptide libraries, the improvements in the screening methods including mass spectrometry MS/MS techniques and the strategies to synthesize OBOC cyclic peptide libraries.

Combinatorial Library Screening Coupled to Mass Spectrometry to Identify Valuable Cyclic Peptides.

Combinatorial library screening coupled to mass spectrometry (MS) analysis is a practical approach to identify useful peptides. Cyclic peptides can have high biological activity, selectivity, and affinity for target proteins, and high stability against proteolytic degradation. Here we describe two strategies to prepare combinatorial libraries suitable for MS analysis to accelerate the discovery of cyclic peptide structures. Both approaches use ChemMatrix resin and the linker 4-hydroxymethylbenzoic acid. One strategy involves the synthesis of a one-bead-two-peptides library in which each bead contains both the cyclic peptide and its linear counterpart to facilitate MS analysis. The other protocol is based on the synthesis of a cyclic depsipeptide library in which a glycolamidic ester group is incorporated by adding glycolic acid. After library screening, the ring is opened and the peptide is released simultaneously for subsequent MS analysis. © 2016 by John Wiley & Sons, Inc.

Friendly strategy to prepare encoded one bead-one compound cyclic peptide library.

One bead-one peptide libraries allow the screening of suitable ligands for any target protein. Short cyclic peptides are ideal ligands for affinity chromatography because of their high affinity and selectivity for the target protein and stability against proteases. We designed a library synthesis strategy to facilitate the identification of cyclic peptides by MS consisting of (a) sequential incorporation of a mixture of Fmoc-Ala-OH and Fmoc-Asp[2-phenylisopropyl (OPp)]-OH (15:85) to Gly-oxymethylbenzamide-ChemMatrix (Gly-HMBA-CM) resin, (b) synthesis of the combinatorial library on the resin by the divide-couple-recombine method, (c) removal of OPp with 4% TFA, (d) peptide cyclization on solid phase through side-chain Asp and amino terminus, and (e) removal of side chain protecting groups with a 95% TFA cocktail. Peptides were cleaved from the beads with ammonia and the linear code was sequenced by MALDI-TOF MS/MS. The high capacity of ChemMatrix resin together with the sensitivity of MS allows code sequencing from a single bead.

Transglycosylation specificity of Acremonium sp. α-rhamnosyl-ß-glucosidase and its application to the synthesis of the new fluorogenic substrate 4-methylumbelliferyl-rutinoside.

Transglycosylation potential of the fungal diglycosidase α-rhamnosyl-ß-glucosidase was explored. The biocatalyst was shown to have broad acceptor specificity toward aliphatic and aromatic alcohols. This feature allowed the synthesis of the diglycoconjugated fluorogenic substrate 4-methylumbelliferyl-rutinoside. The synthesis was performed in one step from the corresponding aglycone, 4-methylumbelliferone, and hesperidin as rutinose donor. 4-Methylumbelliferyl-rutinoside was produced in an agitated reactor using the immobilized biocatalyst with a 16% yield regarding the sugar acceptor. The compound was purified by solvent extraction and silica gel chromatography. MALDI-TOF/TOF data recorded for the [M+Na](+) ions correlated with the theoretical monoisotopic mass (calcd [M+Na](+): 507.44 m/z; obs. [M+Na](+): 507.465 m/z). 4-Methylumbelliferyl-rutinoside differs from 4-methylumbelliferyl-glucoside in the rhamnosyl substitution at the C-6 of glucose, and this property brings about the possibility to explore in nature the occurrence of endo-ß-glucosidases by zymographic analysis.

Ethylenediaminetetraacetic acid (EDTA) as an auxiliary tool in the electrospray ionization mass spectrometry analysis of native and derivatized beta-cyclodextrins, maltoses, and fructans contaminated with Ca and/or Mg.

The effect of Ca(2+) (and Mg(2+)) and the disodium salt of ethylenediaminetetraacetic acid (EDTA), a well known Ca(2+) (and Mg(2+)) chelating agent, on the volatilization/ionization of carbohydrates by using electrospray ionization mass spectrometry has been studied. Model compounds such as maltoses (maltose to maltoheptaose), beta-cyclodextrins (beta-cyclodextrin, methyl-beta-cyclodextrin, heptakis(2,6-di-O-methyl)-beta-cyclodextrin, heptakis(2,3,6-tri-O-methyl)-beta-cyclodextrin, and 2-hydroxypropyl-beta-cyclodextrin) and fructans (sucrose, 1-ketose, nystose, and 1F-fructofuranosylnystose) were used.