Accurate Dereplication of Bioactive Secondary Metabolites from Marine-Derived Fungi by UHPLC-DAD-QTOFMS and a MS/HRMS Library

Abstract

In drug discovery, reliable and fast dereplication of known compounds is essential for identification of novel bioactive compounds. Here, we show an integrated approach using ultra-high performance liquid chromatography-diode array detection-quadrupole time of flight mass spectrometry (UHPLC-DAD-QTOFMS) providing both accurate mass full-scan mass spectrometry (MS) and tandem high resolution MS (MS/HRMS) data. The methodology was demonstrated on compounds from bioactive marine-derived strains of Aspergillus, Penicillium, and Emericellopsis, including small polyketides, non-ribosomal peptides, terpenes, and meroterpenoids. The MS/HRMS data were then searched against an in-house MS/HRMS library of ~1300 compounds for unambiguous identification. The full scan MS data was used for dereplication of compounds not in the MS/HRMS library, combined with ultraviolet/visual (UV/Vis) and MS/HRMS data for faster exclusion of database search results. This led to the identification of four novel isomers of the known anticancer compound, asperphenamate. Except for very low intensity peaks, no false negatives were found using the MS/HRMS approach, which proved to be robust against poor data quality caused by system overload or loss of lock-mass. Only for small polyketides, like patulin, were both retention time and UV/Vis spectra necessary for unambiguous identification. For the ophiobolin family with many structurally similar analogues partly co-eluting, the peaks could be assigned correctly by combining MS/HRMS data and m/z of the [M + Na]+ ions.

A critical review of producers of small lactone mycotoxins: patulin, penicillic acid and moniliformin

“…Chunmei et al, 2013 Penicillium novaezeelandiae Burton, 1949;Burton and Pausacker, 1947;Frisvad and Filtenborg, 1990 Penicillium paneum Boysen et al, 1996;Dombrinck-Kurtzman, 2007;Nielsen et al, 2006Penicillium patulum Anslow et al, 1943Birkinshaw et al, 1943;Chain et al, 1942;Frisvad, 1981 Berestetskii et al, 1975Penicillium roqueforti Bullerman, 1978Cakmakci et al, 2015;Chunmei et al, 2013;Erdogan et al, 2003;Leistner and Pitt, 1977;Malekinejad et al, 2015;Olivigni and Steiman et al, 1989;Müller and Amend, 1997;Paterson et al, 2003;Vismer et al, 1996 Edwards et al, 1999;Goss et al, 1999;Lykakis et al, 2009 and comparison to an authentic standard (Kildgaard et al, 2014). A MS/MS analysis will only yield few fragments for patulin, and thus several other verification tests have to be done to ensure that the metabolite detected is indeed patulin.…”

Four novel Talaromyces species isolated from leaf litter from Colombian Amazon rain forests

“…Three agar plugs from the Talaromyces isolates grown on CYA, MEA, YES and OA at 25°C for 7 days were extracted as described by Smedsgaard (1997). The detected metabolites were identified by comparison with standards according to Kildgaard et al (2014) and Klitgaard et al (2014). Colony texture and ascomata on OA after 2-week incubation.…”

Peptaibol, Secondary‐Metabolite, and Hydrophobin Pattern of Commercial Biocontrol Agents Formulated with Species of the Trichoderma harzianum Complex

“…Separation was done on an Agilent Poroshell 120 phenyl-hexyl column (2.1 Â 150 mm, 2.7 mm) with al inear gradient consisting of H 2 O( A )a nd (MeCN (B)b oth buffered with 20 mmHCOOH,s tarting at 10% B and increasedt o1 00% in 15 min where it was held for 2min. Theflow rate was set to 0.35 ml/min, and temp.to608.The injection volume was 1 ml [ 88]. MS Detection was performed in ESI þ mode on an Agilent 6550 iFunnel QTOF MS equipped with an Agilent Dual JetStream ESI source.Mass spectra were recorded as centroid data from m/z 85 -1700 in MS mode,a nd from m/z 30 -1700 in MS/MS mode.A utomated data-dependent acquisition MS/HRMS (auto-MS/HRMS) analysis was conducteda tt hree distinct fragmentations energies (10,20, and 40 eV) [88].…”

“…Subsequently, the identity of compounds was tentatively verified by MS/HRMS data, searching for fragile bonds and the UV/VIS data [86][87] [88].…”

“…Table 10). All extracts were also scrutinized for mycotoxins (s. s.) known from the genus Trichoderma, viz.t richodermin and harzianum A/B [41],a sw ell as other mycotoxins (s. s.),w hich have not yet been described from the genus Trichoderma such as T-2toxin, gliotoxin, aflatoxins, etc.None of these toxins or other toxic compounds were detected, even though using at riple data analysis approach of the UHPLC-DAD-qTOF data files: i)U V/VIS data were compared to our UV/Vis library [86], ii)full scan data were inspected by the aggressive dereplication approach [87] using the accurate mass and isotopic pattern of the toxins,a sw ell as all other Trichoderma-derived metabolites.F or those compounds with areference standard, the retention time was compared, iii)finally,all MS/HR-MS data were matched against the DTU Mycotoxin-Fungal Secondary Metabolite MS/HRMS library [88],w hich currently contains 1,400 compounds,including all major mycotoxins.Part of the library is freely available to download (277 compounds in Agilent PCDL format)f rom the homepage of the Te chnical University of Denmark [88][89].…”