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 thinlayer chromatography.

The Aspergillus fumigatus Protein GliK Protects against Oxidative Stress and Is Essential for Gliotoxin Biosynthesis

“…4C). RP-HPLC analysis of NaBH 4 -reduced organic extracts, with and without subsequent 5=-IAF alkylation (11,12), from wild-type and mutant cultures indicated the absence of gliotoxin production by the deletion mutant (data not shown). Matrix-assisted laser desorption ionization time-offlight (MALDI-TOF) MS analysis of reduced and alkylated organic extracts from the wild type confirmed the presence of the gliotoxin [i.e., the presence of diacetamidofluorescein-gliotoxin, or GT-(AF) 2 ], while labeled gliotoxin was undetectable in organic extracts from the ⌬gliK 26933 mutant (Fig.…”

“…This was not the case for A. fumigatus ATCC 26933, where the amount of gliotoxin produced was readily detectable prior to NaBH 4 -mediated reduction (11,12), and subsequent reduction confirmed gliotoxin presence by the appearance of the dithiol form at a reduced retention time (3) in organic extracts of the wild-type culture supernatants (data not shown). A method for gliotoxin detection by alkylating the reduced form of gliotoxin with 5=-iodoacetamidofluorescein (5=-IAF) followed by RP-HPLC or MALDI-MS identification (12) provided confirmatory data for gliotoxin production by the A. fumigatus wild-type strain and deficiency in ⌬gliK strains, respectively.…”

“…Extracts were resolubilized in methanol and analyzed using reverse-phase high-performance liquid chromatography (RP-HPLC) and LC-mass spectrometry (LC-MS) as previously described (30,33). Reduction and alkylation (using 5=-iodoacetamidofluorescein; 5=-IAF) of organic extracts was performed as described previously (11,12). For intracellular metabolite analysis, mycelia were lysed as described previously (7,17) prior to reduction and alkylation (11,12).…”

“…Reduction and alkylation (using 5=-iodoacetamidofluorescein; 5=-IAF) of organic extracts was performed as described previously (11,12). For intracellular metabolite analysis, mycelia were lysed as described previously (7,17) prior to reduction and alkylation (11,12). Alternatively, total mycelial thiol quantitation was undertaken using aldrithiol-4 (11 mg/ml in ethanol) analysis (35).…”

Biomarkers for Invasive Aspergillosis: The Challenges Continue

“…There is an interesting perspective on the potential detection of nonribosomal cyclic peptides and depsipeptides by MS as highly specific fungal markers [186] and a recent report describes the derivatization of gliotoxin [187], which is produced by nonribosomal peptide synthesis, to make it detectable by MALDI-TOF MS. The potential of MALDI-TOF using metabolomic approaches is demonstrated by the successful profiling of low-molecular mass volatile compounds in the headspace (the air or empty space left above the contents in a sealed container) of C. albicans urine suspensions by another MS method, proton-transfer reaction MS [188].…”