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

Camperi, Silvia A.; Giudicessi, Silvana L.; Martínez‐Ceron, María C.; Gurevich-Messina, Juan M.; Saavedra, Soledad L.; Acosta, Gerardo; Cascone, Osvaldo; Erra‐Balsells, Rosa; Alberício, Fernando

doi:10.1002/cpch.2

Cited by 7 publications

(3 citation statements)

References 17 publications

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“…The optimized protocol used a high-loading 300 μm polystyrene aminomethyl hydroxymethylbenzoic acid resin. After side chain deprotection, single beads were arrayed into individual tubes and peptides were cleaved using an adapted vapor cleavage protocol with ammonium hydroxide . This protocol allowed for the recovery of chloroalkane-tagged peptide from a single resin bead, without extraction steps, and resolubilization in an aqueous solution that could be used directly in CAPA (Figure A; see Supporting Information for detailed methods).…”

Section: Resultsmentioning

confidence: 99%

Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships

2021

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The efficiency with which polycationic peptides penetrate the cytosol depends on the number and overall patterning of arginine residues. While general trends and unusually penetrant patterns of arginine residues have been discovered, prior work has not effectively leveraged high-throughput screens to measure cytosolic penetration rather than total cell uptake. In this work, we adapted the chloroalkane penetration assay, which exclusively measures cytosolic penetration, to screen peptide libraries in a high-throughput, quantitative, and automation-ready manner. We demonstrate the usefulness of the screening platform by efficiently exploring how the number, patterning, and stereochemistry of arginine residues affect the cytosolic penetration of a model 10-residue peptide.

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Section: Resultsmentioning

confidence: 99%

Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships

2021

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“…For that purpose, the linker 4‐hydroxymethylbenzoic acid (HMBA) was used to introduce a cleavage site between the peptide and the resin . As has been previously reported , the ammonia vapor was easily removed by evaporation to avoid its interference in the MS peptide analysis. The chemical structure of CM, composed entirely of polyethyleneglycol (PEG) monomers that contain exclusively primary ether bonds, made it chemically stable to ammonia thus avoiding sample contamination that would affect MS analysis.…”

Section: Resultsmentioning

confidence: 99%

Simple method to assess stability of immobilized peptide ligands against proteases

et al. 2017

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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.

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“…Also, cyclic peptides can be used as ligands due to their reduced conformational flexibility compared to linear peptides which confers strong resistance to proteolytic degradation. Many approaches to prepare and screen cyclic libraries have been described (Manegatti et al 2013b ; Camperi et al 2016 ) and reviewed by Martínez-Ceron et al ( 2016 ). Also, peptide stability can be increased by synthesizing dendrimer analogs with a polylysine core typically containing four to eight branches.…”

Section: Preliminary Peptide Ligand Evaluationmentioning

confidence: 99%

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

Barredo-Vacchelli

Giudicessi

Martínez‐Ceron

et al. 2021

Int J Pept Res Ther

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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.

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

Cited by 7 publications

References 17 publications

Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships

Parallel Screening Using the Chloroalkane Penetration Assay Reveals Structure-Penetration Relationships

Simple method to assess stability of immobilized peptide ligands against proteases

Peptide Affinity Chromatography Applied to Therapeutic Antibodies Purification

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