Biology subjects

Shepherd, R. A.

Publications and source records attributed to Shepherd, R. A..

4 recordsLinked to original sources

bioRxiv2026

MALDI Tandem Mass Spectrometry for Colony-Based Dereplication of Natural Products

Microbial libraries remain an important resource for natural product discovery; however, constructing taxonomically and chemically diverse collections remains a challenge. Advances in dereplication strategies, including molecular networking, have reduced the rediscovery of known bioactive molecules and facilitated the identification of novel chemical scaffolds, but these approaches are typically applied after library construction or to existing repositories. Furthermore, many dereplication workflows require scaled fermentation and extraction, increasing the time needed to assess a microbes metabolite profile. Here, we integrate matrix-assisted laser desorption/ionization tandem mass spectrometry (MALDI-MS/MS) into the bioinformatics platform IDBac, enabling streamlined characterization of microbial taxonomic identity, metabolite production potential, and preliminary metabolite annotation through GNPS2 molecular networking. This miniaturized high-content workflow facilitates strain prioritization by providing metabolite annotations directly from single microbial colonies prior to scale-up and extraction. Application of this approach to marine actinomycetes enabled the annotation of lavanducyanin and multiple napyradiomycin analogs. Subsequent investigation led to the discovery of napyradiomycin B8 from marine Streptomyces sp. CNZ-289, which was confirmed by 1D and 2D NMR spectroscopy and MALDI-MS/MS. Expanding this workflow to an untargeted analysis of 25 commensal marine vertebrate-derived bacterial isolates resulted in the annotation of several known bioactive natural products, including surugamides, antimycins, desferrioxamine siderophores, and the isolation and elucidation of harmane derivatives using NMR. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=109 SRC="FIGDIR/small/733640v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@c92cfforg.highwire.dtl.DTLVardef@1a9522borg.highwire.dtl.DTLVardef@151b309org.highwire.dtl.DTLVardef@c1531f_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2025

IDBac: an open-access web platform and compendium for the identification of bacteria by MALDI-TOF mass spectrometry.

The identification and analysis of bacteria is central to the microbiological sciences. While gene sequencing methods have been the standard to achieve this, use of MALDI-TOF mass spectrometry (MS), particularly in clinical microbiology, provides high-throughput identification to the subspecies level. However, biotyping has yet to be adopted outside of clinical settings due to the lack of a centralized public database of MS protein signatures that would facilitate isolate identification via spectral comparison. Further, current platforms lack meaningful ways to compare multiple properties from large numbers of bacterial isolates. Herein we present the IDBac web platform, a crowd-sourced central knowledgebase of protein MS signatures of >1400 strains spanning 6 bacterial phyla. Accompanying the knowledgebase is analysis infrastructure to identify unknown isolates, probe relationships within culture collections using metadata integration, and visualize specialized metabolite differences within groups of closely related bacteria. To highlight this utility and encourage wide community contribution, examples of each are presented.

microbiology↗

bioRxiv2025

Development of a High-Throughput Ion Mobility Spectrometry-Based Screening Platform for Kainoid Synthase Biocatalysts

Random mutagenesis generates large, diverse enzyme libraries, however high-throughput screening is challenging when transformations yield isomeric small molecule products. We developed a matrix-assisted laser desorption/ionization-trapped ion mobility spectrometry-mass spectrometry (MALDI-TIMS-MS) platform for rapid, label-free screening of libraries of kainoid synthases, Fe/KG-dependent cyclases that produce the isomeric neurotransmitters kainic acid (KA) and kainic acid lactone (KAL). This platform achieves near-baseline separation of KA and KAL directly from microbial biomass, with parallel reaction monitoring-PASEF (PRM-PASEF) enhancing signal-to-noise for isomeric product ratio analysis. Using this workflow, we screened a 318-variant DsKabC/GfKabC DNA shuffle library in under 30 minutes, identifying seven variants with improved KAL conversion. Two variants showed a near-complete shift toward KAL production with greater substrate consumption, while retaining favorable expression profiles compared to GfKabC. These results establish MALDI-TIMS-MS as a generalizable platform for high-throughput isomeric product screening and provides new kainoid synthase variants to probe structure-function relationships for further development as biocatalysts. Importantly, this screening approach can be broadly applied to interrogate enzyme variants acting on small molecules, delivering rapid and accurate quantitative insights without reliance on chromatography, enzyme purification, or substrate derivatization. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/674781v2_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@185877dorg.highwire.dtl.DTLVardef@18234d6org.highwire.dtl.DTLVardef@15b0df9org.highwire.dtl.DTLVardef@f28f8c_HPS_FORMAT_FIGEXP M_FIG C_FIG

bioengineering↗

bioRxiv2025

MALDI-TIMS-MS2 Imaging and Annotation of Natural Products in Fungal-Bacterial Co-Culture

Mass spectrometry imaging (MSI) is a powerful tool for monitoring the spatial distributions of microbial metabolites directly from culture. MSI can identify secretion and retention patterns for microbial metabolites, allowing for the assessment of chemical communication within complex microbial communities. Microbial imaging via matrix-assisted laser desorption/ionization (MALDI) MSI remains challenging due to high sample complexity and heterogeneity associated with the required sample preparation, making annotation of molecules by MS1 alone challenging. The implementation of trapped ion mobility spectrometry (TIMS) has increased the dimensionality of MALDI-MSI experiments, allowing for the resolution of isomers and isobars, and can increase sensitivity of metabolite detection within complex samples. Parallel reaction monitoring - parallel accumulation serial fragmentation (prm-PASEF) leverages TIMS to enhance the targeted acquisition of MS2 data by increasing the number of precursors that can be fragmented in a single acquisition. Recently, imaging prm-PASEF (iprm-PASEF) has been developed to provide more accurate annotation from MALDI-TIMS-MSI datasets through the inclusion of MS2. Here, we showcase the use of MALDI iprm-PASEF to provide rapid and accurate annotation coproporphyrin III directly from a bacterial-fungal co-culture between Glutamicibacter arilaitensis (strain JB182) and Penicillium solitum (strain #12). Additionally, we present a workflow for untargeted iprm-PASEF precursor selection directly in SCiLS Lab, followed by direct export for iprm-PASEF acquisition. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC="FIGDIR/small/653367v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@5bef36org.highwire.dtl.DTLVardef@1b5f199org.highwire.dtl.DTLVardef@87ff8org.highwire.dtl.DTLVardef@98857_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗