title: Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry.
abstract: Gliotoxin is produced by non-ribosomal peptide synthesis and secreted from certain fungi, including Aspergillus fumigatus. It is an epipolythiodioxopiperazine that contains an intact disulphide bridge and is the focus of intense research as a consequence of its negative immunomodulatory properties. Gliotoxin detection is generally enabled by reversed-phase-high-performance liquid chromatography (RP-HPLC), with absorbance detection (220-280 nm), or liquid chromatography-mass spectrometry, yet detection is not readily achievable by matrix-assisted laser desorption ionisation-time-of-flight mass spectrometry (MALDI-ToF MS). We have developed a single-pot derivatisation strategy which uses sodium borohydride-mediated reduction of gliotoxin followed by immediate alkylation of exposed thiols by 5'-iodoacetamidofluorescein to yield a stable product, diacetamidofluorescein-gliotoxin (GT-(AF)(2)), of molecular mass 1103.931 Da ((M+H)+). This product is readily detectable by RP-HPLC and exhibits a 6.8-fold increase in molar absorptivity compared with gliotoxin, which results in a higher sensitivity of detection (40 ng; 125 pmoL). GT-(AF)(2) also fluoresces (excitation/emission, 492:518 nm). Unlike free gliotoxin, the product (>800 fmol) is detectable by MALDI-ToF MS. Sporidesmin A can also be detected by RP-HPLC and MALDI-ToF MS (>530 fmol) using this strategy. We also demonstrate that the strategy facilitates detection of gliotoxin (mean ± SD = 3.55 ± 0.07 μg 100 μL(-1); n = 2) produced by A. fumigatus, without the requirement for organic extraction of culture supernatants and associated solvent removal. GT-(AF)(2) is also detectable (150 ng; 460 pmol) by thin-layer chromatography.
authors: Department of Biology, National University of Ireland Maynooth, Maynooth, Co. Kildare, Ireland.
authors: Carol
authors: C
authors: Davis
authors: Natasha
authors: N
authors: Gordon
authors: Sinéad
authors: S
authors: Murphy
authors: Ishwar
authors: I
authors: Singh
authors: Kevin
authors: K
authors: Kavanagh
authors: Stephen
authors: S
authors: Carberry
authors: Sean
authors: S
authors: Doyle
mesh_terms: D001232:Aspergillus fumigatus / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D005912:Gliotoxin / Q000032:analysis* / Q000302:isolation & purification; D007166:Immunosuppressive Agents / Q000032:analysis* / Q000302:isolation & purification; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
chemical_list: D007166:Immunosuppressive Agents; D005912:Gliotoxin