Dereplicating and Spatial Mapping of Secondary Metabolites from Fungal Cultures in Situ

Abstract

Ambient ionization mass spectrometry techniques have recently become prevalent in natural product research due to their ability to examine secondary metabolites in situ. These techniques retain invaluable spatial and temporal details that are lost through traditional extraction processes. However, most ambient ionization techniques do not collect mutually supportive data, such as chromatographic retention times and/or UV/vis spectra, and this can limit the ability to identify certain metabolites, such as differentiating isomers. To overcome this, the droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) was coupled with UPLC–PDA–HRMS–MS/MS, thus providing separation, retention times, MS data, and UV/vis data used in traditional dereplication protocols. By capturing these mutually supportive data, the identity of secondary metabolites can be confidently and rapidly assigned in situ. Using the droplet–LMJ–SSP, a protocol was constructed to analyze the secondary metabolite profile of fungal cultures without any sample preparation. The results demonstrate that fungal cultures can be dereplicated from the Petri dish, thus identifying secondary metabolites, including isomers, and confirming them against reference standards. Furthermore, heat maps, similar to mass spectrometry imaging, can be used to ascertain the location and relative concentration of secondary metabolites directly on the surface and/or surroundings of a fungal culture.

Media studies to enhance the production of verticillins facilitated by in situ chemical analysis

“…Previously, a dereplication strategy [67] demonstrated the effectiveness of direct identification of secondary metabolites from cultures grown in Petri dishes. The peak areas of targeted compounds were averaged to build graphs (Fig.…”

“…These cultures were grown on a suite of different solid media, and productivity was assessed by taking advantage of new instrumentation that facilitates the screening of the chemistry of the cultures. The droplet-liquid microjunction-surface sampling probe (droplet probe) permits the in situ analysis of targeted secondary metabolites directly from the surface of cultures [40, 67]. Our goal was to enhance the biosynthesis of verticillins by identifying a strain that produces the highest quantity of the analogues under appropriate fermentation conditions in the shortest period of time.…”

Ambient Mass Spectrometry Imaging Using Direct Liquid Extraction Techniques

“…51 LMJ-SSP has been used for the analysis of molecules separated on TLC plates, 47 tissue profiling, 55,56 and spatial mapping of metabolites in microbial 57 and fungal communities. 58 …”

In situ mass spectrometry monitoring of fungal cultures led to the identification of four peptaibols with a rare threonine residue

“…These small peptides have been reported to possess a wide variety of bioactivities, such as antimicrobial (Figueroa et al, 2013), anthelmintic (Schiell et al, 2001; Thirumalachar, 1968), and cytotoxic (He et al, 2006). As part of a multidisciplinary program to identify new anticancer leads from nature (Ayers et al, 2012; Figueroa et al, 2013; Kinghorn et al, 2016), a protocol was implemented to monitor the chemistry of fungal cultures in situ using a recently described droplet–liquid microjunction–surface sampling probe (droplet–LMJ–SSP) coupled to an ultraperformance liquid chromatography–high resolution mass spectrometry (UPLC–HRMS) system (Kertesz and Van Berkel, 2010; Paguigan et al, 2016; Sica et al, 2016b; Sica et al, 2015; Sica et al, 2016c). …”

“…This system is designed to perform microextractions on the surface of a sample and directly inject the extract into LC-MS (Kertesz and Van Berkel, 2010, 2013, 2014). Recently, this technique has been applied towards in situ analyses of fungal and plant metabolites with various goals, such as dereplication of fungal cultures (Sica et al, 2015), spatial and temporal mapping of the biosynthesis of targeted leads in fungal cultures (Paguigan et al, 2016; Sica et al, 2016b; Sica et al, 2016c), and the characterization of complex mixtures in a suite of plant tissues (Sica et al, 2016a). A major benefit of this technique is the ability to monitor the biosynthesis of fungal secondary metabolites, both with respect to space and time, without having to extract the entire culture.…”