title: Development of immobilized-pepsin microreactors coupled to nano liquid chromatography and tandem mass spectrometry for the quantitative analysis of human butyrylcholinesterase.
abstract: Human butyrylcholinesterase is a serine hydrolase that reacts with organophosphorus compounds (OP) to form stable adducts. These adducts are valuable biomarkers for OP exposure and can be analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) after a preliminary digestion step in solution. However, this digestion step is time-consuming and cannot be directly coupled with LC-MS set ups. Therefore, the aim of this work was to develop pepsin-based immobilized enzyme microreactors (IMERs) for the rapid digestion of human butyrylcholinesterase (HuBuChE). Various IMERs were synthesized by grafting different amounts of pepsin on a CNBr-sepharose gel and the grafting yield was measured by a bicinchoninic acid assay (BCA). A sensitive nanoLC-MS/MS method was developed to evaluate the digestion yields of HuBuChE on IMERs which was made possible by a synthetic peptide which was used as a calibrant. The digestion was optimized by studying the impact of different parameters such as the digestion time, the temperature and the amount of pepsin grafted on IMER. This optimization allowed HuBuChE to be digested with-in 20min without pretreatment and with digestion yields up to 20%. The repeatability of the IMER synthesis and HuBuChE digestion was highlighted with the characterization of 3 similar IMERs. Finally, the digestion yields of HuBuChE were higher with IMERs when compared to a typical in solution digestion.
authors: Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM), UMR CBI 8231, ESPCI Paris, PSL Research University, 10 rue Vauquelin, Paris, France.
authors: Maud
authors: M
authors: Bonichon
authors: Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM), UMR CBI 8231, ESPCI Paris, PSL Research University, 10 rue Vauquelin, Paris, France.
authors: Audrey
authors: A
authors: Combès
authors: DGA, CBRN Defence, 5 rue Lavoisier, Vert-le-Petit, France.
authors: Charlotte
authors: C
authors: Desoubries
authors: DGA, CBRN Defence, 5 rue Lavoisier, Vert-le-Petit, France.
authors: Anne
authors: A
authors: Bossée
authors: Department of Analytical, Bioanalytical Sciences and Miniaturization (LSABM), UMR CBI 8231, ESPCI Paris, PSL Research University, 10 rue Vauquelin, Paris, France; UPMC, Paris Sorbonne Universités, 4 place Jussieu, Paris, France. Electronic address: valerie.pichon@espci.fr.
authors: Valérie
authors: V
authors: Pichon
mesh_terms: D000818:Animals; D019149:Bioreactors; D002091:Butyrylcholinesterase / Q000032:analysis* / Q000378:metabolism*; D002138:Calibration; D002853:Chromatography, Liquid / Q000379:methods*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D036103:Nanotechnology / Q000379:methods*; D009943:Organophosphorus Compounds / Q000737:chemistry / Q000378:metabolism; D010434:Pepsin A / Q000737:chemistry / Q000378:metabolism*; D010446:Peptide Fragments / Q000032:analysis / Q000378:metabolism; D013552:Swine; D053719:Tandem Mass Spectrometry / Q000379:methods*; D013997:Time Factors
chemical_list: D004800:Enzymes, Immobilized; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D002091:Butyrylcholinesterase; D010434:Pepsin A
keywords: HuBuChE; Immobilized enzyme reactor; NanoLC–MS/MS; Pepsin digestion