title: Optimization of a nano-enzymatic reactor for on-line tryptic digestion of polypeptide conjugates by capillary electrophoresis.
abstract: This work aims at studying the optimization of an on-line capillary electrophoresis (CE)-based tryptic digestion methodology for the analysis of therapeutic polypeptides (PP). With this methodology, a mixture of surrogate peptide fragments and amino acid were produced on-line by trypsin cleavage (enzymatic digestion) and subsequently analyzed using the same capillary. The resulting automation of all steps such as injection, mixing, incubation, separation and detection minimizes the possible errors and saves experimental time. In this paper, we first study the differents parameters influencing PP cleavage inside the capillary (plug length, reactant concentration, incubation time, diffusion and electrophoretic plugs mixing). In a second part, the optimization of the electrophoretic separation conditions of generated hydrolysis products (nature, pH and ionic strength (I) of the background electrolyte (BGE)) is described. Using the optimized conditions, excellent repeatability was obtained in terms of separation (migration times) and proteolysis (number of products from enzymatic hydrolysis and corresponding amounts) demonstrating the robustness of the proposed methodology.
authors.affiliation: Institut des Biomolécules Max Mousseron-IBMM, Centre National de la Recherche Scientifique, Université de Montpellier, Unité Mixte de Recherche 5247, Faculté de Pharmacie, Montpellier cedex 5, France.
authors.forename: Yoann
authors.initials: Y
authors.lastname: Ladner
authors.affiliation: Institut des Biomolécules Max Mousseron-IBMM, Centre National de la Recherche Scientifique, Université de Montpellier, Unité Mixte de Recherche 5247, Faculté de Pharmacie, Montpellier cedex 5, France.
authors.forename: Gaelle
authors.initials: G
authors.lastname: Coussot
authors.affiliation: Institut des Biomolécules Max Mousseron-IBMM, Centre National de la Recherche Scientifique, Université de Montpellier, Unité Mixte de Recherche 5247, Faculté de Pharmacie, Montpellier cedex 5, France.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Ebner
authors.affiliation: Institut des Biomolécules Max Mousseron-IBMM, Centre National de la Recherche Scientifique, Université de Montpellier, Unité Mixte de Recherche 5247, Faculté de Pharmacie, Montpellier cedex 5, France.
authors.forename: Amal
authors.initials: A
authors.lastname: Ibrahim
authors.affiliation: GemacBio, Lieu-dit Berganton, Saint-Jean d'Illac, France.
authors.forename: Laetitia
authors.initials: L
authors.lastname: Vidal
authors.affiliation: Institut des Biomolécules Max Mousseron-IBMM, Centre National de la Recherche Scientifique, Université de Montpellier, Unité Mixte de Recherche 5247, Faculté de Pharmacie, Montpellier cedex 5, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Perrin
mesh_terms: D008735:5-Methoxytryptamine / Q000032:analysis / Q000737:chemistry; D000818:Animals; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000295:instrumentation*; D004867:Equipment Design; D006868:Hydrolysis; D009994:Osmolar Concentration; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry; D010455:Peptides / Q000032:analysis* / Q000737:chemistry; D011107:Polylysine / Q000032:analysis / Q000737:chemistry; D014357:Trypsin / Q000737:chemistry*
chemical_list: D010446:Peptide Fragments; D010455:Peptides; D011107:Polylysine; D008735:5-Methoxytryptamine; D014357:Trypsin
keywords: Capillary electrophoresis; On-line trypsin digestion; Proteolysis product separation; Trypsin nanoreactor