GNPS - nanoRAPIDS, an analytical pipeline for the discovery of novel bioactive metabolites in complex culture extracts at nanoscale

Here we present an analytical platform for the efficient identification and prioritization of low abundance bioactive compounds at nanoliter scale, called nanoRAPIDS. NanoRAPIDS encompasses analytical scale separation and nanofractionation of natural extracts, followed by the bioassay of interest, automated mass spectrometry identification, and Global Natural Products Social molecular networking (GNPS) for dereplication. As little as 10 microliters crude extract is thereby fractionated into 384 fractions. First, bioactive congeners of iturins and surfactins were identified in Bacillus, based on their activity against Gram-positive bacteria, Gram-negative bacteria and/or fungi. Subsequently, bioactive molecules were identified in an extensive network of angucyclines elicited by catechol in cultures of Streptomyces sp. This allowed the discovery of a highly unusual N-acetylcysteine conjugate of saquayamycin, despite low production levels in an otherwise abundant molecular family. These data underline the utility and broad application of the technology for the prioritization of minor bioactive compounds in complex extracts.