• Home
  • Search Results
  • 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.

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.

Rapid communications in mass spectrometry : RCM (2014-08-19)
Aleksander Salwiński, David Da Silva, Raphaël Delépée, Benoît Maunit
ABSTRACT

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. 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). 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. 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.

MATERIALS
Product Number
Brand
Product Description

Supelco
Ammonium formate, eluent additive for LC-MS, LiChropur, ≥99.0%
Sigma-Aldrich
Ammonium formate, reagent grade, 97%
Sigma-Aldrich
Ammonium formate, ≥99.995% trace metals basis
Sigma-Aldrich
L-Tyrosine, reagent grade, ≥98% (HPLC)
Sigma-Aldrich
Ammonium formate, BioUltra, ≥99.0% (calc. based on dry substance, NT)
Sigma-Aldrich
Ammonium formate solution, BioUltra, 10 M in H2O
Supelco
(+)-Catechin, analytical standard
Sigma-Aldrich
L-Tyrosine, from non-animal source, meets EP, USP testing specifications, suitable for cell culture, 99.0-101.0%
Sigma-Aldrich
L-Tyrosine, BioUltra, ≥99.0% (NT)
Supelco
L-Tyrosine, Pharmaceutical Secondary Standard; Certified Reference Material
SAFC
L-Tyrosine
USP
L-Tyrosine, United States Pharmacopeia (USP) Reference Standard
Sigma-Aldrich
L-Tyrosine, FG
Supelco
L-Tyrosine, certified reference material, TraceCERT®
(±)-Catechin hydrate, primary reference standard
Sigma-Aldrich
3-(2,4-Dihydroxyphenyl)propionic acid, ≥95.0% (HPLC)