title: Supported inhibitor for fishing lipases in complex biological media and mass spectrometry identification.
abstract: A synthetic phosphonate inhibitor designed for lipase inhibition but displaying a broader range of activity was covalently immobilized on a solid support to generate a function-directed tool targeting serine hydrolases. To achieve this goal, straightforward and reliable analytical techniques were developed, allowing the monitoring of the solid support's chemical functionalization, enzyme capture processes and physisorption artifacts. This grafted inhibitor was tested on pure lipases and serine proteases from various origins, and assayed for the selective capture of lipases from several complex biological extracts. The direct identification of captured enzymes by mass spectrometry brought the proof of concept on the efficiency of this supported covalent inhibitor. The features and limitations of this "enzyme-fishing" proteomic tool provide new insight on solid-liquid inhibition process.
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France; CNRS, Aix Marseille Université, Centrale Marseille, iSm2 UMR 7313, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
authors: Vincent
authors: V
authors: Delorme
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors: Brigitt
authors: B
authors: Raux
authors: CNRS, Aix Marseille Université, Institut de Microbiologie de la Méditerranée FR3479, plate-forme Marseille Protéomique (MaP), 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors: Rémy
authors: R
authors: Puppo
authors: CNRS, Aix Marseille Université, Centrale Marseille, iSm2 UMR 7313, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France. Electronic address: julien.leclaire@univ-lyon1.fr.
authors: Julien
authors: J
authors: Leclaire
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors: Jean-François
authors: JF
authors: Cavalier
authors: CNRS, Aix Marseille Université, Centrale Marseille, iSm2 UMR 7313, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
authors: Sylvain
authors: S
authors: Marc
authors: CNRS, Aix Marseille Université, Centrale Marseille, iSm2 UMR 7313, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
authors: Pavan-Kumar
authors: PK
authors: Kamarajugadda
authors: CNRS, Aix Marseille Université, Centrale Marseille, iSm2 UMR 7313, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
authors: Gérard
authors: G
authors: Buono
authors: CNRS, Aix Marseille Université, Centrale Marseille, iSm2 UMR 7313, avenue Escadrille Normandie-Niemen, 13397 Marseille Cedex 20, France.
authors: Frédéric
authors: F
authors: Fotiadu
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France. Electronic address: canaan@imm.cnrs.fr.
authors: Stéphane
authors: S
authors: Canaan
authors: CNRS, Aix-Marseille Université, Enzymologie Interfaciale et Physiologie de la Lipolyse, UMR 7282, 31 Chemin Joseph Aiguier, 13402 Marseille Cedex 20, France.
authors: Frédéric
authors: F
authors: Carrière
mesh_terms: D000595:Amino Acid Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000138:chemical synthesis* / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D007700:Kinetics; D008049:Lipase / Q000037:antagonists & inhibitors* / Q000235:genetics / Q000378:metabolism; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry / Q000379:methods*; D008956:Models, Chemical; D008969:Molecular Sequence Data; D015394:Molecular Structure
chemical_list: D004791:Enzyme Inhibitors; D008049:Lipase
keywords: Complex mixtures; Grafted inhibitors; Lipases; Mass spectrometry; Phosphonates