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Marine sponges and their associated symbionts produce a structurally diverse and complex set of natural products including alkaloids, terpenoids, peptides, lipids, and steroids. A single sponge with its symbionts can produce all of the above-mentioned classes of molecules and their analogs. Most approaches to evaluating sponge chemical diversity have focused on major metabolites that can be isolated and characterized; therefore, a comprehensive evaluation of intra- (within a molecular family; analogs) and inter-chemical diversity within a single sponge remains incomplete. We use a combination of metabolomics tools, including a supervised approach via manual library search and literature search, and an unsupervised approach via molecular networking and MS2LDA analysis to describe the intra and inter-chemical diversity present in Smenospongia aurea. Furthermore, we use imaging mass spectrometry to link this chemical diversity to either the sponge or the associated cyanobacteria. Using these approaches, we identify 7 more molecular features that represent analogs of four previously known peptide/polyketide smenamides and assign the biosynthesis of these molecules to the symbiotic cyanobacteria by imaging mass spectrometry. We extend this analysis to a wide diversity of molecular classes including indole alkaloids and meroterpenes.

Microcapillary electrophoresis (μCE) enables high-resolution separations in miniaturized, automated microfluidic devices. Pairing this powerful separation technique with laser-induced fluorescence (LIF) enables a highly sensitive, quantitative, and compositional analysis of organic molecule monomers and short polymers, which are essential, ubiquitous components of life on Earth. Improving methods for their detection has applications to multiple scientific fields, particularly those related to medicine, industry, and space science. Here, a modular benchtop system using μCE with LIF detection was constructed and tested by analyzing standard amino acid samples of valine, serine, alanine, glycine, glutamic acid, and aspartic acid in multiple borate buffered solutions of increasing concentrations from 10 mM to 50 mM, all pH 9.5. The 35 mM borate buffer solution generated the highest resolution before Joule heating dominated. The limits of detection of alanine and glycine using 35 mM borate buffer were found to be 2.12 nM and 2.91 nM, respectively, comparable to other state-of-the-art μCE-LIF instruments. This benchtop system is amenable to a variety of detectors, including a photomultiplier tube, a silicon photomultiplier, or a spectrometer, and currently employs a spectrometer for facile multi-wavelength detection. Furthermore, the microdevice is easily exchanged to fit the desired application of the system, and optical components within the central filter cube can be easily replaced to target alternative fluorescent dyes. This work represents a significant step forward for the analysis of small organic molecules and biopolymers using μCE-LIF systems.

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