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Comparison of the molecular networks of the WT SH-C52 strain and its mutants followed by peptidogenomic analysis allowed us to confirm that we had detected the thanamycin molecule that, despite application of several different cultivation and extraction procedures, eluded detection by traditional approaches. A possible reason why thanamycin was initially missed by conventional approaches was because thanamycin is produced transiently and in low quantities compared with the rest of the molecules observed in the sample. The discovery of thanamycin was made possible by using nanoDESI techniques, and this exemplifies that our methodology allows for the detection of the proverbial needle in the haystack.

Conclusion

By adapting nanoDESI MS for profiling live microbial colonies grown on Petri dishes, we have demonstrated that it is possible to perform highly sensitive metabolic profiling directly off living microbial communities without the need for chemical tags, labels, or any sample preparation whatsoever. This ability to capture a wide variety of molecular classes within a single mass spectrum directly from a live specimen will prove extremely useful in visualizing the “big picture” of these signal transduction networks and will allow researchers to see a more complete chemotype, resulting in more accurate correlations to their observed phenotype. We have also shown that analysis of the MS/MS data by using molecular networking enables detection and visualization of related compounds via spectral relationships within and between data sets as chemical families are grouped together. In addition, MS/MS networks enable dereplication ( Inexpensive Sale With Mastercard MaxMara Virgin WoolBlend HighRise Pants Free Shipping Authentic Free Shipping Cheap Quality Purchase Cheap Price r6CjhDRN
) (i.e., finding “known unknowns”) and allow for prioritization of the analysis of individual MS/MS signals by finding variation/diversity of molecules across multiple conditions/species even before its identity is determined, as we have shown for detection and partial characterization of thanamycin. Tools such as nanoDESI and molecular networking by spectral alignment are an important addition to our effort to create a “Rosetta stone” for microbial interactions through observing and classifying metabolic exchange ( Footaction Cheap Online Fake Sale Online Lott Isoli Embroidered Hoodie Sweatshirt Ganni Really Cheap Shoes Online 2018 Unisex Sale Online Outlet Locations Online EDPjfEYk
). In a more general context, the methods described here constitute a powerful set of tools in systems (micro)biology for investigating the spatiotemporal dynamics of diverse metabolic exchange processes.

Preparation of Bacterial Samples.

Colonies of A3(2) were prepared by inoculating 1 μL of harvested spores onto ISP2 nutrient agar (7.5 g agar, 5 g malt extract, 2 g yeast extract, and 2 g dextrose in 500 mL milli-Q water) and incubating at 30 °C until the desired time point. Colonies of 3610/PY79, ES129, PAO1, and sp. strain SH-C52 were prepared by inoculating 1 μL of cell stock into 4 mL of LB broth and incubating at 30 °C until an OD of 0.4 was reached. From this starter culture, 0.2 to 1.0 μL was inoculated onto ISP2 nutrient agar and allowed to grow at 30 °C until the desired time point. Colonies of MC2 were prepared by inoculating 1 μL of cell stock into 4 mL of LB broth and incubating at 30 °C until an OD of 0.4 was reached. From this starter culture, 1.0 μL was inoculated onto LB nutrient agar and allowed to grow at 30 °C until the desired time point. For the data shown, , , , , and were analyzed by nanoDESI at 72, 48, 36, 48, and 42 h after inoculation, respectively.

Cocultures of PY79 and A3(2) were prepared by inoculating two 1-μL colonies of onto ISP2 nutrient agar ∼5 mm apart and allowing to grow for 18 h at 30 °C, to fortify the colony, after which 0.2 μL of PY79 was inoculated 1 mm from the terminal end of one of the colonies and allowed to grow for an additional 36 h at 30 °C.

NanoDESI Instrument Setup.

Data collection was performed by using two different nanoDESI ionization sources of similar design in two different laboratories. The first was located in the Laskin laboratory at Pacific Northwest National Laboratory and was coupled to a Thermo LTQ-Orbitrap mass spectrometer equipped with collision-induced dissociation capabilities. The second nanoDESI source was located in the Dorrestein laboratory at the University of California, San Diego, and was coupled to a Thermo LTQ-FT-ICR MS also capable of collision-induced dissociation. The overall design of both instruments, as illustrated in slash neck logo sweater Black Vivienne Westwood Sale Free Shipping 61i0QB
, is very simple in that solvent is loaded into a 250- or 500-μL syringe, which is placed in a syringe pump. Solvent is initially pushed through 300 μm o.d. × 100 μm i.d. fused silica capillary tubing to a stainless steel union in which voltage is applied and the tubing is stepped down to 150 μm o.d. × 50 μm i.d., which continues to the liquid bridge, where it meets the self-aspirating nanospray capillary. The position of both the primary and the nanospray capillaries are controlled by two separate xyz manual positioners and monitored by mounted video cameras. All analyses were performed in positive ion mode in the mass range of from 100 to 2,000. Both the primary and the nanospray capillaries were 150 μm o.d. × 50 μm i.d., with solvent being delivered and removed from the liquid bridge at approximate 45° angles. The spray voltage was kept between 2.0 and 3.0 kV depending on the solvent, with the solvents largely being acetonitrile/0.05% formic acid in water (1:1) or acetonitrile:toluene:methanol (35:15:50) running at a flow rate of 0.8 to 2.5 μL/min. The droplet size using this configuration was ∼200 μm in diameter.

NanoDESI Profiling and Time-Course Experiments.

Profiling and time-course experiments were performed by using a simple protocol because absolutely no sample preparation is needed for analysis. For each measurement, samples were removed from the incubator, photographed, and placed directly onto the nanoDESI sample stage for analysis. By using a series of mounted cameras for guidance, the sample was manually raised until the liquid bridge came into contact with the sample surface ( Movie S1 ). Signal from the sample surface was produced almost immediately, and, depending on the nature of the sample and analyte, remained consistent enough at a single location to usually allow for 20-min acquisitions of data dependent tandem mass spectra. For time-course experiments, the solvent droplet was only in contact with the colony for 20 s for each measurement, after which the sample was placed back in the incubator until the following time point. For experiments in which voltage was not used, the flow rate of the nanoDESI was stopped before bringing the sample to the probe, the droplet was allowed to sit on the colony surface for 20 s, the sample was removed, and the flow rate and voltage were turned back on for data collection. Control samples were treated exactly the same as samples that were analyzed by using the nanoDESI whereby they were removed from the incubator at each time point, photographed, left outside the incubator while the samples were analyzed, and then placed back into the incubator without being analyzed by nanoDESI themselves.

Construction of Molecular MS/MS Networks.

Tandem mass spectra were clustered with MS-Cluster ( Valentino Striped ButtonUp Blazer Store Sale 2018 New Quality Outlet Store Discount Enjoy Clearance 2018 Unisex BDl1WtN
) to group repeatedly acquired spectra from the same molecules into cluster-consensus spectra with a higher signal-to-noise ratio. As the obtained spectra contained fragmentation signatures for not only peptides, but also polysaccharides, lipids, small-molecular metabolites, and even small proteins, the following nondefault settings were used to avoid bias toward peptide scoring: clustering model LTQ_TRYP, minimum spectrum quality 0 (to avoid peptide-specific spectrum quality metrics), disabled assign-charges and correct-pm commands. Cluster-consensus spectra were processed by applying square root transforms to fragment peak intensities (to increase/decrease the influence of low/high intensity peaks, respectively), scaled to Euclidian norm 1 and used for the construction of molecular MS/MS networks in two steps: () pairwise spectral alignment to find pairs of spectra from related molecules and () selection of significant pairwise alignments to define the MS/MS network. For each pair of spectra S and S′, spectral alignment was computed by defining modification mass as follows:

With the difference between their precursor masses and by finding matching fragment peaks between S and S′ as follows: () peaks p∈S and p′∈S′ are eligible matches if |mz(p) − mz(p′)| ≤t or if |mz(p) + M − mz(p′)| ≤t, for a predetermined fragment tolerance t; () match scores between matching peaks are defined as the product of their normalized peak intensities; () peak matches define a bipartite matching problem of selecting the highest scoring subset of matching peaks whereby each peak is matched to at most one peak in the other spectrum (a classical computer science problem with well known algorithms for finding optimal solutions). As a result of the spectrum intensity scaling and peak match scores, the optimal solution of each bipartite matching problem corresponds to the highest possible cosine between the intensities of matched peaks ( Rani Arabella Long Sleeve Cashmere Sweater Newest Cheap Online cjIMj
). Pairs of cluster-consensus spectra were considered for spectral alignment if their molecular masses differed by less than 45% and as much as 400 Da. Each spectrum retained only as many as 10 highest-cosine alignments and pair wise alignments with cosine ≥0.7 and ≥6 matched peaks were used to define the MS/MS networks ( Michelle Mason KneeLength Striped Skirt Buy Cheap Very Cheap rQk5B
) whereby each node is a cluster-consensus spectrum and each edge corresponds to a significant pair wise alignment. All algorithms assumed precursor mass tolerance of 1.0 Da and fragment mass tolerance of 0.3 Da.

Acknowledgments

The authors thank Peter Tonge (Stony Brook University) for providing the strain. Work in this area in the P.C.D. laboratory for the development of real-time MS was supported by National Institutes of Health (NIH) Grant GM094802; work on interspecies interaction was supported by NIH Grant AI095125 and Johnson Johnson; work in this area in the N.B. laboratory was supported by NIH Grant 1-P41-RR024851; and work in this area in the K.P. laboratory is supported by NIH grant AI095125. This work was also supported in part by NIH Grant GM085770 (to B.S.M.); the German Research Foundation, Deutsche Forschungsgemeinschaft (DFG), Grant DFG-FOR854 GR2673/2-1 (to H.G.); the Chemical Imaging Program at Pacific Northwest National Laboratory (PNNL) (P.R. and J.L.); the US Department of Energy (DOE) Science Undergraduate Laboratory Internship (SULI) program at PNNL (B.S.H.); Dutch Science Organization Ecology Regarding Gene-Modified Organisms Grant838.06.101 (to M.v.d.V. and J.M.R.); and Netherlands Genomics Initiative (NGI) ECOLINC and PreSeed (M.v.d.V. and J.M.R.). A part of the research described in this paper was performed at the W. R. Wiley Environmental Molecular Sciences Laboratory, a national scientific user facility sponsored by the DOE’s Office of Biological and Environmental Research and located at PNNL, which is operated by Battelle for the DOE.

Footnotes

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Present address: Roach and Associates, Seymour, WI 54165.

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