Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

Abstract

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac-a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

Addition of insoluble fiber to isolation media allows for increased metabolite diversity of lab-cultivable microbes derived from zebrafish gut samples

“…Library isolates were prepared for bioinformatics analysis using IDBac, a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry pipeline that allows for rapid analysis of microbial proteins and specialized metabolites. 17,20 The standard IDBac workflow uses a 70% aqueous formic acid solution to lyse cells but we found that this procedure was not efficient at lysing the more mucoid bacterial colonies isolated. 20 A trifluoroacetic acid (TFA) extraction prior to plating samples for analysis was the most effective method to consistently collect viable MS fingerprint profiles for IDBac analysis.…”

“…17,20 The standard IDBac workflow uses a 70% aqueous formic acid solution to lyse cells but we found that this procedure was not efficient at lysing the more mucoid bacterial colonies isolated. 20 A trifluoroacetic acid (TFA) extraction prior to plating samples for analysis was the most effective method to consistently collect viable MS fingerprint profiles for IDBac analysis. 21 Measuring proteins in the 3,000 to 15,000 Da range, IDBac organized the microbial isolates into a pseudo-phylogeny dendrogram based on MS fingerprint protein similarity.…”

“…The IDBac pipeline also generates metabolite association networks (MANs) for rapid visualization of metabolite production and allows for a simple comparison between bacterial isolates grown in different conditions. 20 Figure 3A shows a dendrogram highlighting 16 isolates that were grown on ISP2 agar, commonly used for lab cultivation, or SNF + fiber agar plates. 22 In a MAN, nodes correspond to ions detected in a sample and the relationships between production across strains or conditions are visualized as arrows connecting nodes.…”

“…The IDBac workflow and software were designed to rapidly collect protein and specialized metabolite profiles of microbial libraries produced from environmental samples utilizing a MALDI-TOF mass spectrometer. 17,20 Of the 118 isolates, 82 were examined using the IDBac workflow to organize the microbial library ( Figure 2 ).The remaining 36 isolates were not analyzed due to either poor data quality collection during MALDI-TOF MS analysis or failure to recultivate from the initial glycerol stock ( Table S1 ). Based on the MS protein fingerprints of the isolates, the newly constructed library represents three phyla.…”

Addition of insoluble fiber to isolation media allows for increased metabolite diversity of lab-cultivable microbes derived from zebrafish gut samples

“…IDBac analysis reveals that the addition of insoluble fiber supports an equally diverse cultivation of gut microbes as traditionally nutrient-rich media Library isolates were prepared for bioinformatics analysis using IDBac, a matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry pipeline that allows for rapid analysis of microbial proteins and secondary metabolites. 15,17 The standard IDBac workflow uses a 70% aqueous formic acid solution to lyse cells but we found that this procedure was not efficient at lysing the more mucoid bacterial colonies isolated. 17 A trifluoroacetic acid (TFA) extraction prior to plating samples for analysis was the most effective method to consistently collect viable MS fingerprint profiles for IDBac analysis.…”