title: The use of enzyme-coupled magnetic nanoparticles for studying the spectra of unusual substrates of mushroom tyrosinase by direct surface-assisted laser desorption/ionisation and high-resolution electrospray ionisation quadrupole-quadrupole-time-of-flight mass spectrometry.
abstract: BACKGROUND
  Tyrosinase-coupled magnetic particles (EMPs) were used to demonstrate that resorcinol-containing tyrosinase inhibitors are oxidised by tyrosinase only in the presence of the enzyme's classic substrate. This shows the potential for the application of EMPs as a non-organic matrix for monitoring enzymatic conversion of a novel substrate family directly on-the-spot, principally due to minimal enzyme requirement per analysis.
  
  
  METHODS
  Tyrosinase was covalently coupled to core-shell-type silica-coated iron oxide magnetic nanoparticles (EMPs) that were applied as non-organic SALDI matrix suitable for studying low-mass compounds using a classic matrix-assisted laser desorption/ionisation time-of-flight (MALDI-TOF) mass spectrometer. Because of the dual function of the EMPs - enzyme host and non-organic matrix - we describe this ionisation method as Enzyme-coupled Nanoparticles-Assisted LDI-MS (ENALDI-MS). Supplementary studies of the enzymatic conversion of glabridin and 3-(2,4-dihydroxyphenyl)propionic acid (DHPA) were conducted by high-resolution electrospray ionisation quadrupole-quadrupole-time-of-flight mass spectrometry (ESI-QqTOF-MS).
  
  
  RESULTS
  The initial experiment involving EMPs as non-organic matrix (ENALDI-MS) showed enzymatic conversion of glabridin, a strong tyrosinase inhibitor, only in the presence of L-Tyr, the classic tyrosinase substrate. These findings were evaluated by ESI-QqTOF-MS proving that glabridin and DHPA are converted into the corresponding quinones by tyrosinase only in the presence of the auxiliary monophenol or o-diphenol substrates (L-Tyr and catechin, respectively) capable of regenerating the active site of tyrosinase.
  
  
  CONCLUSIONS
  EMPs were shown to be useful as a non-organic matrix to monitor enzymatic conversion of the novel tyrosinase substrate family directly on-the-spot with a minimal enzyme consumption (6.5 pmol/spot). Results obtained by ENALDI-MS were fully confirmed by ESI-QqTOF-MS demonstrating that resorcinol-containing tyrosinase inhibitors may be oxidised by the enzyme in the presence of its classic substrates.
authors.affiliation: Normandy University, UCBN, UR ABTE EA 4651, F-14032, Caen, France.
authors.forename: Aleksander
authors.initials: A
authors.lastname: Salwiński
authors.forename: David
authors.initials: D
authors.lastname: Da Silva
authors.forename: Raphaël
authors.initials: R
authors.lastname: Delépée
authors.forename: Benoît
authors.initials: B
authors.lastname: Maunit
mesh_terms: D000363:Agaricales / Q000201:enzymology*; D004800:Enzymes, Immobilized / Q000737:chemistry / Q000378:metabolism*; D058185:Magnetite Nanoparticles / Q000737:chemistry*; D014442:Monophenol Monooxygenase / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D012118:Resorcinols / Q000737:chemistry / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
chemical_list: D004800:Enzymes, Immobilized; D058185:Magnetite Nanoparticles; D012118:Resorcinols; D014442:Monophenol Monooxygenase; C031389:resorcinol