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    Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry


    Davis, Carol and Gordon, Natasha and Murphy, Sinead and Singh, Ishwar and Kavanagh, Kevin and Carberry, Stephen and Doyle, Sean (2011) Single-pot derivatisation strategy for enhanced gliotoxin detection by HPLC and MALDI-ToF mass spectrometry. Analytical and Bioanalytical Chemistry, 401 (8). pp. 2519-2529. ISSN 1618-2642

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    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.

    Item Type: Article
    Additional Information: The definitive version of this article is available at DOI 10.1007/s00216-011-5344-1
    Keywords: Epipolythiodioxopiperazine; Gliotoxin; NRPS; Redox; Sporidesmin A;
    Academic Unit: Faculty of Science and Engineering > Biology
    Item ID: 4151
    Depositing User: Dr. Kevin Kavanagh
    Date Deposited: 30 Jan 2013 16:56
    Journal or Publication Title: Analytical and Bioanalytical Chemistry
    Publisher: Springer Verlag
    Refereed: Yes
    URI:

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