Advances in structure elucidation of small molecules using mass spectrometry

The structural elucidation of small molecules using mass spectrometry plays an important role in modern life sciences and bioanalytical approaches. This review covers different soft and hard ionization techniques and figures of merit for modern mass spectrometers, such as mass resolving power, mass accuracy, isotopic abundance accuracy, accurate mass multiple-stage MS(n) capability, as well as hybrid mass spectrometric and orthogonal chromatographic approaches. The latter part discusses mass spectral data handling strategies, which includes background and noise subtraction, adduct formation and detection, charge state determination, accurate mass measurements, elemental composition determinations, and complex data-dependent setups with ion maps and ion trees. The importance of mass spectral library search algorithms for tandem mass spectra and multiple-stage MS(n) mass spectra as well as mass spectral tree libraries that combine multiple-stage mass spectra are outlined. The successive chapter discusses mass spectral fragmentation pathways, biotransformation reactions and drug metabolism studies, the mass spectral simulation and generation of in silico mass spectra, expert systems for mass spectral interpretation, and the use of computational chemistry to explain gas-phase phenomena. A single chapter discusses data handling for hyphenated approaches including mass spectral deconvolution for clean mass spectra, cheminformatics approaches and structure retention relationships, and retention index predictions for gas and liquid chromatography. The last section reviews the current state of electronic data sharing of mass spectra and discusses the importance of software development for the advancement of structure elucidation of small molecules

Analysis and Interpretation of Mass Spectrometry Imaging Datasets

Mass spectrometry imaging (MSI) has recently emerged as a widely used technique in application areas including pharmaceutical evaluation, ecosystem physiology, high-throughput screening and many others. Mass spectrometry images are made by raster scanning a sample in two dimensions and acquiring mass spectra at each location. Although there are many ways of performing MSI and a diversity of suitable samples, data analysis steps are generally consistent across experiments. These standard analysis steps include access to and management of the raw data, processing of individual spectra with centroiding and denoising algorithms. They also include normalization approaches to both improve spot-to-spot variability and quantification of molecules. Finally, multivariate statistics approaches are used to navigate the complexity of sometimes overwhelming large volume of information. Here, we present all of these analyses approaches and discuss how and why they are used in MSI

Phosphate Availability Modulates Root Exudate Composition and Rhizosphere Microbial Community in a Teosinte and a Modern Maize Cultivar

Domestication and breeding have affected interactions between plants and their microbiomes in ways that are only beginning to be understood but may have important implications for recruitment of rhizosphere microorganisms, particularly under stress conditions. We investigated the responses of a modern maize (Zea mays subsp. mays) cultivar and its wild relative, teosinte (Z. mays subsp. parviglumis), to different phosphate availabilities. We appraised responses of the plant-microbial holobiont to phosphate stresses by profiling root exudate metabolomes, and microbial communities in the root endosphere and rhizosphere. We also performed plate assays to quantify phosphate-solubilizing microorganisms from the rhizosphere. Although root exudate metabolite profiles were distinct between the teosinte and modern maize under high phosphate, both plants shifted exudate compositions in response to phosphate stress toward a common metabolite profile. Root and rhizosphere microbial communities also responded significantly to both plant type and the phosphate availability. A subset of bacterial and fungal taxa were differentially abundant under the different phosphate conditions, with each of the three conditions favoring different taxa. Both teosinte and maize rhizospheres harbored phosphate-solubilizing microorganisms under all growth conditions. These results suggest that the root exudation response to phosphate stress was conserved through the domestication of maize from teosinte, shifting exudation levels of specific metabolites. Although microbial communities also shifted, plate-based assays did not detect selective recruitment of phosphate solubilizers in response to phosphate availability

A novel method to evaluate nutrient retention by biological soil crust exopolymeric matrix

Aims: Biological soil crusts (biocrusts) are microbial communities commonly found in the upper layer of arid soils. These microorganisms release exopolysaccharides (EPS), which form the exopolymeric matrix (EPM), allowing them to bond soil particles together and survive long periods of dryness. The aim of this work is to develop methods for measuring metabolite retention by biocrust EPM and EPS. Methods: We report new methods for the investigation of metabolite sorption on biocrusts compared to the underlying unconsolidated subcrust fraction. A 13C–labeled bacterial lysate metabolite mixture was incubated with biocrust, subcrust and biocrust-extracted EPS. Non-sorbed metabolites were extracted and analyzed by liquid chromatography/mass spectrometry. Results: This simple and rapid approach enabled the comparison of metabolite sorption on the biocrust EPM or EPS versus mineral sorption on the underlying soils. Our results suggest that the biocrust (and its extracted EPS) sorb more metabolites, especially amino acids and organic acids, than the underlying subcrust. Conclusions: This study demonstrates a useful method to highlight the essential role of biocrust (especially the EPM), which acts as a passive nutrient filter, sequestering metabolites released by microbes during wetting events. This may facilitate recovery of the community upon wetting and further enhance biocrust survival and nutrient retention

Flux balance modeling to predict bacterial survival during pulsed-activity events

Desert biological soil crusts (BSCs) are cyanobacteria-dominated surface soil microbial communities common to plant interspaces in arid environments. The capability to significantly dampen their metabolism allows them to exist for extended periods in a desiccated dormant state that is highly robust to environmental stresses. However, within minutes of wetting, metabolic functions reboot, maximizing activity during infrequent permissive periods. iMicrocoleus vaginatus/i, a primary producer within the crust ecosystem and an early colonizer, initiates crust formation by binding particles in the upper layer of soil via exopolysaccharides, making microbial dominated biological soil crusts highly dependent on the viability of this organism. Previous studies have suggested that biopolymers play a central role in the survival of this organism by powering resuscitation, rapidly forming compatible solutes, and fueling metabolic activity in dark, hydrated conditions. To elucidate the mechanism of this phenomenon and provide a basis for future modeling of BSCs, we developed a manually curated, genome-scale metabolic model of iMicrocoleus vaginatus/i (iNJ1153). To validate this model, gas chromatography-mass spectroscopy (GC-MS) and liquid chromatography-mass spectroscopy (LC-MS) were used to characterize the rate of biopolymer accumulation and depletion in in hydrated iMicrocoleus vaginatus/i under light and dark conditions. Constraint-based flux balance analysis showed agreement between model predictions and experimental reaction fluxes. A significant amount of consumed carbon and light energy is invested into storage molecules glycogen and polyphosphate, while iβ/i-polyhydroxybutyrate may function as a secondary resource. Pseudo-steady-state modeling suggests that glycogen, the primary carbon source with the fastest depletion rate, will be exhausted if iM. vaginatus /i experiences dark wetting events 4 times longer than light wetting events

Flux balance modeling to predict bacterial survival during pulsed-activity events

Desert biological soil crusts (BSCs) are cyanobacteria-dominated surface soil microbial communities common to plant interspaces in arid environments. The capability to significantly dampen their metabolism allows them to exist for extended periods in a desiccated dormant state that is highly robust to environmental stresses. However, within minutes of wetting, metabolic functions reboot, maximizing activity during infrequent permissive periods. iMicrocoleus vaginatus/i, a primary producer within the crust ecosystem and an early colonizer, initiates crust formation by binding particles in the upper layer of soil via exopolysaccharides, making microbial dominated biological soil crusts highly dependent on the viability of this organism. Previous studies have suggested that biopolymers play a central role in the survival of this organism by powering resuscitation, rapidly forming compatible solutes, and fueling metabolic activity in dark, hydrated conditions. To elucidate the mechanism of this phenomenon and provide a basis for future modeling of BSCs, we developed a manually curated, genome-scale metabolic model of iMicrocoleus vaginatus/i (iNJ1153). To validate this model, gas chromatography-mass spectroscopy (GC-MS) and liquid chromatography-mass spectroscopy (LC-MS) were used to characterize the rate of biopolymer accumulation and depletion in in hydrated iMicrocoleus vaginatus/i under light and dark conditions. Constraint-based flux balance analysis showed agreement between model predictions and experimental reaction fluxes. A significant amount of consumed carbon and light energy is invested into storage molecules glycogen and polyphosphate, while iβ/i-polyhydroxybutyrate may function as a secondary resource. Pseudo-steady-state modeling suggests that glycogen, the primary carbon source with the fastest depletion rate, will be exhausted if iM. vaginatus /i experiences dark wetting events 4 times longer than light wetting events