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 microbe's 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