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

Abstract

For decades, researchers have lacked the ability to rapidly correlate microbial identity with bacterial metabolism. Since specialized metabolites are critical to bacterial function and survival in the environment, we designed a data acquisition and bioinformatics technique (IDBac) that utilizes in situ matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to analyze protein and specialized metabolite spectra of single bacterial colonies from agar plates. We demonstrated the power of our approach by discriminating between two Bacillus subtilis colonies in under 30 minutes, which differ by a single genomic mutation, solely on the basis of their differential ability to produce cyclic peptide antibiotics surfactin and plipastatin. Next, we employed our IDBac technique to detect subtle intra-species differences in the production of metal scavenging acyl-desferrioxamines in a group of eight freshwater Micromonospora isolates that share >99% sequence similarity in the 16S rRNA gene. Finally, we employed our method to simultaneously extract protein and specialized metabolite MS profiles from unidentified species of Lake Michigan sponge-associated bacteria cultivated on an agar plate. In just 3 hours, we created hierarchical protein MS groupings of 11 environmental isolates (10 MS replicates each, for a total of 110 samples) that accurately mirrored phylogenetic groupings. We further distinguished isolates within these groupings, which share nearly identical 16S rRNA gene sequence identity, based on inter- and intra-species differences in specialized metabolite production. To our knowledge, IDBac is the first attempt to couple in situ MS analyses of protein content and specialized metabolite production to allow the distinction of closely related bacterial colonies.SignificanceMass spectrometry is a powerful technique that has been used to identify bacteria via protein content, and to assess bacterial function in an environment via analysis of specialized metabolites. However, until now these analyses have operated independently, and this has resulted in the inability to rapidly connect bacterial phylogenetic identity with patterns of specialized metabolism. To bridge this gap, we designed a MALDI-TOF mass spectrometry data acquisition and bioinformatics pipeline (IDBac) to discriminate both intact protein and specialized metabolite spectra directly from bacterial cells grown on agar. To our knowledge, this is the first technique that organizes bacteria into highly similar phylogenetic groups and allows for comparison of metabolic differences of hundreds of isolates in just a few hours.

Whole Cell MALDI Fingerprinting Is a Robust Tool for Differential Profiling of Two-Component Mammalian Cell Mixtures

“…Minimal sample processing circumvents complex and time-consuming experimental steps necessary for cell proteomic analyses. MALDI protein spectral profiles generated during analyses are species specific and serve as unique cellular ID fingerprints [19][20][21]. Speed, simplicity, sensitivity and specificity for large biomolecules in complex matrices confer whole cell MALDI fingerprinting as a robust, facile and inexpensive technique.…”

Biodiversity, Bioactivity, and Metabolites of High Desert Derived Oregonian Soil Bacteria

“…Prof. Brian Murphy and team recently developed IDBac, a pipeline to simplify the bacterial prioritization process for chemical screening [10,11] . One feature is the hierarchical clustering based on MALDI‐TOF‐MS protein spectra, offering a different approach to assess the taxonomic diversity of bacterial colonies [12] .…”

“…In collaboration with Prof Brian Murphy (UIC), bacterial colonies were subjected to their IDBac pipeline [10,11] . For matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) analysis, the bacteria were streaked on M2 agar plates individually and grown for 12 days.…”

Spatial Analyses of Specialized Metabolites: The Key to Studying Function in Hosts

“…Our lab is optimizing and conducting IMS on squid hatchlings inoculated with V. fischeri strains to determine differences in the chemistry produced between strains and the spatial distribution of the molecules produced within the host tissue. Additionally, we are in the process of characterizing these bacterial strains using our novel MALDI-TOF MS-based workflow that allows for rapid proteomic and metabolomic classification ( 13 ). We hope to gain a better understanding of the symbiotic relationship between the squid and V. fischeri and how colonization in this model system may translate to similar host-microbe interactions.…”