RESUMEN
Nanospray desorption electrospray ionization (nano-DESI) coupled with high-resolution mass spectrometry (MS) and tandem mass spectrometry (MS/MS) enable detailed molecular characterization of living bacterial colonies directly from nutrient agar. The ability to detect molecular signatures of living microbial communities is important for investigating metabolic exchange between species without affecting the viability of the colonies. We describe the protocol for bacterial growth, sample preparation, ambient profiling, and data analysis of microbial communities using nano-DESI MS.
Asunto(s)
Bacterias/crecimiento & desarrollo , Bacterias/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Bacterias/química , Diseño de Equipo , Consorcios Microbianos , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
An automated platform has been developed for acquisition and visualization of mass spectrometry imaging (MSI) data using nanospray desorption electrospray ionization (nano-DESI). The new system enables robust operation of the nano-DESI imaging source over many hours by precisely controlling the distance between the sample and the nano-DESI probe. This is achieved by mounting the sample holder onto an automated XYZ stage, defining the tilt of the sample plane, and recalculating the vertical position of the stage at each point. This approach is useful for imaging of relatively flat samples such as thin tissue sections. Custom software called MSI QuickView was developed for visualization of large data sets generated in imaging experiments. MSI QuickView enables fast visualization of the imaging data during data acquisition and detailed processing after the entire image is acquired. The performance of the system is demonstrated by imaging rat brain tissue sections. Low background noise enables simultaneous detection of lipids and metabolites in the tissue section. High-resolution mass analysis combined with tandem mass spectometry (MS/MS) experiments enabled identification of the observed species. In addition, the high dynamic range (>2000) of the technique allowed us to generate ion images of low-abundance isobaric lipids. A high-spatial resolution image was acquired over a small region of the tissue section revealing the distribution of an abundant brain metabolite, creatine, on the boundary between the white and gray matter. The observed distribution is consistent with the literature data obtained using magnetic resonance spectroscopy.
Asunto(s)
Automatización , Encéfalo/anatomía & histología , Nanotecnología , Programas Informáticos , Animales , Nanotecnología/instrumentación , Ratas , Espectrometría de Masa por Ionización de Electrospray/instrumentaciónRESUMEN
Reactive nanospray desorption electrospray ionization (nano-DESI) combined with high-resolution mass spectrometry was utilized for the analysis of secondary organic aerosol produced through ozonolysis of limonene (LSOA). Previous studies have shown that LSOA constituents are multifunctional compounds containing at least one aldehyde or ketone groups. In this study, we used the selectivity of the Girard's reagent T (GT) toward carbonyl compounds to examine the utility of reactive nano-DESI for the analysis of complex organic mixtures. In these experiments, 1-100 µM GT solutions were used as the working solvents for reactive nano-DESI analysis. Abundant products from the single addition of GT to LSOA constituents were observed at GT concentrations in excess of 10 µM. We found that LSOA dimeric and trimeric compounds react with GT through a simple addition reaction resulting in formation of the carbinolamine derivative. In contrast, reactions of GT with monomeric species result in the formation of both the carbinolamine and the hydrazone derivatives. In addition, several monomers did not react with GT on the time scale of our experiment. These molecules were characterized by relatively high values of the double bond equivalent and low oxygen content. Furthermore, because addition of a charged GT tag to a neutral molecule eliminates the discrimination against the low proton affinity compounds in the ionization process, reactive nano-DESI analysis enables quantification of individual compounds in the complex mixture. For example, we were able to estimate for the first time the amounts of dimers and trimers in the LSOA mixture. Specifically, we found that the most abundant LSOA dimer was detected at the ~0.5 pg level and the total amount of dimers and trimers in the analyzed sample was ~11 pg. Our results indicate that reactive nano-DESI is a valuable approach for examining the presence of specific functional groups and for the quantification of compounds possessing these groups in complex mixtures.
Asunto(s)
Nanotecnología/métodos , Compuestos Orgánicos/análisis , Compuestos Orgánicos/química , Espectrometría de Masa por Ionización de Electrospray/métodos , Aerosoles , Betaína/análogos & derivados , Betaína/química , Ciclohexenos/química , Limoneno , Ozono/química , Terpenos/químicaRESUMEN
Integrating the governing chemistry with the genomics and phenotypes of microbial colonies has been a "holy grail" in microbiology. This work describes a highly sensitive, broadly applicable, and cost-effective approach that allows metabolic profiling of live microbial colonies directly from a Petri dish without any sample preparation. Nanospray desorption electrospray ionization mass spectrometry (MS), combined with alignment of MS data and molecular networking, enabled monitoring of metabolite production from live microbial colonies from diverse bacterial genera, including Bacillus subtilis, Streptomyces coelicolor, Mycobacterium smegmatis, and Pseudomonas aeruginosa. This work demonstrates that, by using these tools to visualize small molecular changes within bacterial interactions, insights can be gained into bacterial developmental processes as a result of the improved organization of MS/MS data. To validate this experimental platform, metabolic profiling was performed on Pseudomonas sp. SH-C52, which protects sugar beet plants from infections by specific soil-borne fungi [R. Mendes et al. (2011) Science 332:1097-1100]. The antifungal effect of strain SH-C52 was attributed to thanamycin, a predicted lipopeptide encoded by a nonribosomal peptide synthetase gene cluster. Our technology, in combination with our recently developed peptidogenomics strategy, enabled the detection and partial characterization of thanamycin and showed that it is a monochlorinated lipopeptide that belongs to the syringomycin family of antifungal agents. In conclusion, the platform presented here provides a significant advancement in our ability to understand the spatiotemporal dynamics of metabolite production in live microbial colonies and communities.
Asunto(s)
Bacillus subtilis/metabolismo , Espectrometría de Masas/métodos , Mycobacterium smegmatis/metabolismo , Pseudomonas aeruginosa/metabolismo , Streptomyces coelicolor/metabolismo , Bacillus subtilis/genética , Secuencia de Bases , Familia de Multigenes , Mycobacterium smegmatis/genética , Pseudomonas aeruginosa/genética , Streptomyces coelicolor/genéticaRESUMEN
Ambient ionization imaging mass spectrometry is uniquely suited for detailed spatially resolved chemical characterization of biological samples in their native environment. However, the spatial resolution attainable using existing approaches is limited by the ion transfer efficiency from the ionization region into the mass spectrometer. Here, we present a first study of ambient imaging of biological samples using nanospray desorption ionization (nano-DESI). Nano-DESI is a new ambient pressure ionization technique that uses minute amounts of solvent confined between two capillaries comprising the nano-DESI probe and the solid analyte for controlled desorption of molecules present on the substrate followed by ionization through self-aspirating nanospray. We demonstrate highly sensitive spatially resolved analysis of tissue samples without sample preparation. Our first proof-of-principle experiments indicate the potential of nano-DESI for ambient imaging with a spatial resolution of better than 12 µm. The significant improvement of the spatial resolution offered by nano-DESI imaging combined with high detection efficiency will enable new imaging mass spectrometry applications in clinical diagnostics, drug discovery, molecular biology, and biochemistry.
Asunto(s)
Espectrometría de Masa por Ionización de Electrospray/métodos , Animales , Encéfalo/metabolismo , Humanos , Riñón/metabolismo , Nanotecnología , RatasRESUMEN
Mass Spectrometric Imaging (MSI) allows the generation of 2D ion density maps that help visualize molecules present in sections of tissues and cells. The combination of spatial resolution and mass resolution results in very large and complex data sets. New capabilities are necessary for efficient analysis and interpretation of this data. This work details the development and application of the capability to process, visualize, query, and analyze spatial mass spectrometry data. Applications include the generation of 2D maps for selected spectra, the manipulation of the heat maps, and the identification of spectral peaks. Heat maps are generated by projecting the sum of intensity vs. time spectra of each pixel for selected m/z value or range. These capabilities take the form of a new interactive software toolkit, MSI QuickView. This software approach is a significant advance over the previous state-of-the art methods that required the conversion of the RAW data using one software, manual assembly of the data, and visualization in another software.