Miniaturized affinity chromatography: A powerful technique for the isolation of high affinity GAGs sequences prior to their identification by MALDI-TOF MS.

Autor: Jeanroy F; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Institut des Sciences Analytiques, UMR, 5280, 5 Rue de la Doua, F-69100, Villeurbanne, France., Comby-Zerbino C; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Institut Lumière Matière, UMR 5306, F-69100, Villeurbanne, France., Demesmay C; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Institut des Sciences Analytiques, UMR, 5280, 5 Rue de la Doua, F-69100, Villeurbanne, France., Dugas V; Université de Lyon, CNRS, Université Claude Bernard Lyon 1, Institut des Sciences Analytiques, UMR, 5280, 5 Rue de la Doua, F-69100, Villeurbanne, France. Electronic address: vincent.dugas@univ-lyon1.fr.
Jazyk: angličtina
Zdroj: Analytica chimica acta [Anal Chim Acta] 2023 Oct 09; Vol. 1277, pp. 341656. Date of Electronic Publication: 2023 Jul 25.
DOI: 10.1016/j.aca.2023.341656
Abstrakt: Glycosaminoglycans (GAGS) are involved in many biological processes through interactions with a variety of proteins, including proteases, growth factors, cytokines, chemokines and adhesion molecules. Identifying druggable GAG-protein interactions for therapeutic purposes is a challenge for the analytical community. In this context, this work investigates the use of a new miniaturized monolithic affinity column (poly(GMA-co-MBA) grafted with antithrombin III (AT III)) to specifically capture and elute high affinity sequences contained in low molecular weight heparin (enoxaparin) for further on-line characterization. This miniaturized, high binding capacity affinity column allows the specific capture of high-affinity oligosaccharide chains from Enoxaparin, even at low concentrations and with a minimal consumption of AT III. In addition to purification, this elution process enables preconcentration for direct analysis by capillary zone electrophoresis. It was found that many of oligosaccharide chains in enoxaparin were eliminated and that certain chain sequences were retained and enriched. Direct coupling with MALDI-TOF MS was successfully used to further characterize the specifically retained oligosaccharides where nano-ESI-TOF MS failed. After optimization of the sample preparation and ionization parameters, direct on-line analysis was performed by applying the elution volume released from the miniaturized affinity column (≤1 μL) directly to the MALDI plate. Finally, this original miniaturized analytical workflow coupling miniaturized AT III-affinity chromatography to MALDI-TOF MS detection is able to select, enrich and detect and identify high affinity sequences (mainly DP4 in size length with a high degree of sulfation) from low molecular weight heparin samples. A more specific selection of GAG sequences can be achieved by increasing the ionic strength during the washing step of affinity chromatography. This is consistent with the known binding pattern between heparin and AT III.
Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
(Copyright © 2023 Elsevier B.V. All rights reserved.)
Databáze: MEDLINE
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