Peptide Affinity Chromatography Based on Combinatorial Strategies for Protein Purification

Camperi, Silvia A.; Martínez‐Ceron, María C.; Giudicessi, Silvana L.; Marani, Mariela M.; Alberício, Fernando; Cascone, Osvaldo

doi:10.1007/978-1-62703-977-2_22

Cited by 21 publications

(22 citation statements)

References 30 publications

(24 reference statements)

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“…4-Hydroxymethylbenzoic acid (HMBA) [6] was used as the linker in order to introduce a cleavage site to release the peptides from the beads before MS analysis. The model peptide H-Phe-Lys-Phe-ArgTyr-Thr-Ala-His-Ser-Gly-Ala-Ser-Gly-NH2 with known trypsin and chymotrypsin cleavage sites [7] was synthesized on HMBA-CM resin (0.63 mmol/g) by using the Fmoc strategy as we have previously described [1]. Ser-Gly-Ala-Ser-Gly was incorporated as a spacer arm at the C-terminal.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Stability Evaluation of Immobilized Peptides Towards Proteases by Mass Spectrometry

Giudicessi¹,

Salum²,

Martínez‐Ceron³

et al. 2015

Peptides 2015, Proceedings of the 24th American Peptide Symposium

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Section: Resultsmentioning

confidence: 99%

“…Short peptides are widely used as ligands in affinity chromatography purification of proteins [1,2]. However, peptidases and proteases present in the crude sample may degrade immobilized peptides, shortening the affinity support useful life.…”

Section: Introductionmentioning

confidence: 99%

Stability Evaluation of Immobilized Peptides Towards Proteases by Mass Spectrometry

Giudicessi¹,

Salum²,

Martínez‐Ceron³

et al. 2015

Peptides 2015, Proceedings of the 24th American Peptide Symposium

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“…Released peptides were eluted from each bead by adding 10 μl acetic acid (AcOH)/acetonitrile (CH 3 CN)/H 2 O (3/4/3) to each micro tube [1] (Scheme 1).…”

Section: Depsipeptide Ring Opening and Peptide Releasementioning

confidence: 99%

“…Short peptides with affinity for target proteins are widely used in therapeutic and diagnostic applications and as ligands in affinity chromatography [1][2][3][4][5][6]. Particularly, cyclic peptides have a great interest because of their resistance to enzymatic degradation and higher selectivity than their linear counterparts [7,8].…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2014

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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.

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“…Short peptides (between 5 and 12 amino acids) have been described as useful ligands for AC because of their low cost, easy chemical synthesis, and higher stability in comparison to protein ligands. Furthermore, the peptide‐based chromatographic supports are more robust during elution and regeneration than protein‐based affinity chromatographic supports that use monoclonal antibodies as ligands …”

Section: Introductionmentioning

confidence: 99%

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

Messina

Giudicessi

Ceron

et al. 2018

Journal of Peptide Science

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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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Peptide Affinity Chromatography Based on Combinatorial Strategies for Protein Purification

Cited by 21 publications

References 30 publications

Stability Evaluation of Immobilized Peptides Towards Proteases by Mass Spectrometry

Stability Evaluation of Immobilized Peptides Towards Proteases by Mass Spectrometry

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

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

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