Coupling MALDI-TOF mass spectrometry protein and specialized metabolite analyses to rapidly discriminate bacterial function

Abstract

SignificanceMass spectrometry is a powerful technique that has been used to identify bacteria by their protein content and to assess bacterial functional traits through analysis of their specialized metabolites. However, until now these analyses have operated independently, which has resulted in the inability to rapidly connect bacterial phylogenetic identity with potential environmental function. To bridge this gap, we designed a MALDI-TOF mass spectrometry data acquisition and bioinformatics pipeline (IDBac) to integrate data from both intact protein and specialized metabolite spectra directly from bacterial cells grown on agar. This technique organizes bacteria into highly similar phylogenetic groups and allows for comparison of metabolic differences of hundreds of isolates in just a few hours.

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.…”

“…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. 17 Figure 2 highlights 82 of the 118 bacterial isolates from our library. 16S rRNA sequencing was performed on a subset of isolates.…”

“…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.…”

“…Though the zebrafish gut microbiome varies slightly throughout development, once adulthood is reached, the gut microbiome is heavily conserved and strongly colonized by γ-Proteobacteria, Firmicutes, and Fusobacteria. 15,16 Using the high-throughput bioinformatics pipeline IDBac, 17 we isolated and analyzed 118 individual colonies from the zebrafish gut microbiome. IDBac analysis showed that our cultivation technique generated a taxonomically diverse library, reflecting that of previous metagenomic studies of the zebrafish gut microbiome, vide supra .…”

LC‐MS/MS‐based metabolic profiling of Escherichia coli under heterologous gene expression stress

“…8,9 However, there is a paucity of information about the metabolic profiles of genetically resistant bacterial populations. 13,14 The data need further screening against METLIN database. 11,12 However, mass-spectrometric data sets depend on CAMERA for compound spectra extraction and annotation.…”