This work was supported by the National Institutes of Health (NIH) National Institute of General Medical Sciences (NIGMS) under award GM138660. J.T.S. was supported by the Charles and Monica Burkett Family Summer Fellowship from the University of Florida. J.P.Z. was supported by NIH grants K22AI7220 (NIAID) and R35GM138369 (NIGMS). A.B.S. was supported by the Cell and Molecular Biology Training Grant at the University of Pennsylvania (T32GM07229).

NOTE: Animal experiments were approved by the Animal Care and Use Committees of the Children&#39;s Hospital of Philadelphia and the University of Pennsylvania Perelman School of Medicine (protocols IAC 18-001316 and 806279).
CAUTION: Clostridium difficile (C. difficile) and Enterococcus faecalis (E. faecalis) are BSLII pathogens and should be handled with extreme caution. Utilize proper decontamination protocols when necessary.
<str...

<ol>
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During MALDI imaging mass spectrometry, it is important to have a flat sample surface to provide for a consistent focal diameter of the incident MALDI laser on the sample substrate. Deviations in sample height can cause the MALDI laser beam to shift out of focus, causing alterations in beam diameter and intensity, which can affect MALDI ionization efficiency. These alterations in ionization efficiency can result in differences in analyte intensity across the tissue surface that are not reflective of the underlying tissue...

The human microbiome is a highly dynamic ecosystem involving molecular interactions of bacteria, viruses, archaea, and other microbial eukaryotes. While microbial relationships have been intensely studied in recent years, much remains to be understood about microbial processes at the chemical level1,2. This is in part due to the unavailability of tools capable of accurately measuring complex microbial environments. Advances in the field of imaging mass spectrometry (IMS) over the past decade have enabled in situ and label-free spatial mapping of many metabolites, lipids, and proteins in biological substrates3,4. Matrix-assisted laser desorption/ionization (MALDI) has emerged as the most common ionization technique used in imaging mass spectrometry, involving the use of a UV laser to ablate material from the surface of a thin tissue section for measurement by mass spectrometry4. This process is facilitated by the application of a chemical matrix applied homogeneously to the surface of the sample, allowing for sequential measurements to be made in a raster pattern across the sample surface. Heat maps of analyte ion intensities are then generated following data acquisition. Recent advances in ionization sources and sampling techniques have enabled the analysis of non-traditional substrates such as bacterial5 and mammalian6,7,8 cellular specimens grown on nutrient agar. The molecular spatial information afforded by IMS can provide unique insight into the biochemical communication of microbe-microbe and host-microbe interactions during infection9,10,11,12,13,14.
Upon Clostridioides difficile infection (CDI), C. difficile is exposed to a rapidly changing microbial environment in the gastrointestinal tract, where polymicrobial interactions are likely to impact infection outcomes15,16. Surprisingly, little is known about the molecular mechanisms of interactions between C. difficile and resident microbiota during infection. For example, enterococci are a class of opportunistic commensal pathogens in the gut-microbiome and have been associated with increased susceptibility to and severity of CDI17,18,19,20. However, little is known about the molecular mechanisms of the interactions between these pathogens. To visualize small-molecule communication between these members of the gut microbiome, bacterial macrocolonies were grown herein on agar to simulate microbe-microbe interactions and&#160;bacterial biofilm formation in a controlled environment. However, obtaining representative metabolic distributions upon MALDI imaging mass spectrometry analysis of bacterial culture specimens is challenging due to the high water content and uneven surface topography of these samples. This is largely caused by the highly hydrophilic nature of agar and the non-uniform agar surface response during moisture removal.
The high water content of agar can also make it challenging to achieve homogeneous MALDI matrix coating and can interfere with subsequent MALDI analysis performed in vacuo21. For example, many MALDI sources operate at pressures of 0.1-10 Torr, which is a sufficient vacuum to remove moisture from the agar and can cause deformation of the sample. These morphological changes in the agar induced by the vacuum environment cause bubbling and cracking in the dried agar material. These artifacts reduce the adherence of the agar to the slide and can cause dismounting or flaking of the sample into the instrument vacuum system. The thickness of the agar samples can be up to 5 mm off the slide, which can create insufficient clearance from ion optics inside the instrument, causing contamination and/or damage to instrument ion optics. These cumulative effects can result in reductions of ion signal reflective of the surface topography, rather than the underlying microbial biochemical interactions. Agar samples must be homogeneously dried and strongly adhered to a microscope slide prior to analysis in vacuo.
This paper demonstrates a sample preparation workflow for the controlled drying of bacterial culture macrocolonies grown on agar media. This multi-step, slower drying process (relative to those previously reported) ensures that the agar will dehydrate uniformly while minimizing the effects of bubbling or cracking of agar samples mounted on microscope slides. By using this gradual drying method, samples are strongly adhered to the microscope slide and amenable for subsequent matrix application and MALDI analysis. This is exemplified using model bacterial colonies of C. difficile grown on agar and murine tissue models harboring CDI with and without the presence of commensal and opportunistic pathogen,&#160;Enterococcus faecalis. MALDI imaging mass spectrometry analyses of both bacterial and tissue models allow for the spatial mapping of amino acid metabolite profiles, providing novel insight into bioenergetic microbial metabolism and communication.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.2 &#956;m Titan3 nylon syringe filters</td>
			<td>Thermo Scientific</td>
			<td>42225-NN</td>
			<td></td>
		</tr>
		<tr>
			<td>1,5-diaminonaphthalene MALDI matrix</td>
			<td>Sigma Aldrich</td>
			<td>2243-62-1</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL Henke Ject luer lock syringes</td>
			<td>Henke Sass Wolf</td>
			<td>4200.000V0&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>275i series convection vacuum gauge</td>
			<td>Kurt J. Lesker company</td>
			<td>KJL275807LL</td>
			<td></td>
		</tr>
		<tr>
			<td>7T solariX FTICR mass spectrometer equipped with a Smartbeam II Nd:YAG MALDI laser system (2 kHz, 355 nm)&#160;</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Acetic acid solution, suitable for HPLC</td>
			<td>Sigma Aldrich</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetonitrile, suitable for HPLC, gradient grade, &#8805;99.9%</td>
			<td>Sigma Aldrich</td>
			<td>75-05-8</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium hydroxide solution, 28% NH3 in H2O, &#8805;99.99% trace metals basis</td>
			<td>Sigma Aldrich</td>
			<td>1336-21-6</td>
			<td></td>
		</tr>
		<tr>
			<td>Autoclavable biohazard bags: 55 gal</td>
			<td>Grainger</td>
			<td>45TV10</td>
			<td></td>
		</tr>
		<tr>
			<td>Biohazard specimen transport bags (8 x 8 in.)</td>
			<td>Fisher Scientific</td>
			<td>01-800-07</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain heart infusion broth</td>
			<td>BD Biosciences</td>
			<td>90003-040</td>
			<td></td>
		</tr>
		<tr>
			<td>C57BL/6 male mice&#160;</td>
			<td>Jackson Laboratories</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CanoScan 9000F Mark II photo and document scanner</td>
			<td>Canon</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>CM 3050S research cryomicrotome</td>
			<td>Leica Biosystems</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Desiccator cabinet</td>
			<td>Sigma Aldrich</td>
			<td>Z268135</td>
			<td></td>
		</tr>
		<tr>
			<td>Diamond tip scriber, Electron Microscopy Sciences&#160;</td>
			<td>Fisher Scientific</td>
			<td>50-254-51</td>
			<td></td>
		</tr>
		<tr>
			<td>Drierite desiccant pellets</td>
			<td>Drierite</td>
			<td>21005</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethanol, 200 Proof</td>
			<td>Decon Labs</td>
			<td>2701</td>
			<td></td>
		</tr>
		<tr>
			<td>flexImaging software</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ftmsControl software</td>
			<td>Bruker Daltonics</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Glass vacuum trap</td>
			<td>Sigma Aldrich</td>
			<td>Z549460</td>
			<td></td>
		</tr>
		<tr>
			<td>HTX M5 TM robotic sprayer</td>
			<td>HTX Technologies</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Indium Tin Oxide (ITO)-coated microscope slides</td>
			<td>Delta Technologies</td>
			<td>CG-81IN-S115</td>
			<td></td>
		</tr>
		<tr>
			<td>In-line HEPA filter to vacuum pump</td>
			<td>LABCONCO</td>
			<td>7386500</td>
			<td></td>
		</tr>
		<tr>
			<td>Methanol, HPLC Grade</td>
			<td>Fisher Chemical</td>
			<td>&#160; 67-56-1</td>
			<td></td>
		</tr>
		<tr>
			<td>MTP slide-adapter II</td>
			<td>Bruker Daltonics</td>
			<td>235380</td>
			<td></td>
		</tr>
		<tr>
			<td>Optimal cutting temperature (OCT) compound</td>
			<td>Fischer Scientific</td>
			<td>23-730-571</td>
			<td></td>
		</tr>
		<tr>
			<td>Peridox RTU Sporicide, Disinfectant and Cleaner</td>
			<td>CONTEC</td>
			<td>CR85335&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>PTFE (Teflon) printed slides, Electron Microscopy Sciences</td>
			<td>VWR</td>
			<td>100488-874</td>
			<td></td>
		</tr>
		<tr>
			<td>Rotary vane vacuum pump RV8</td>
			<td>Edwards</td>
			<td>A65401903</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek Accu-Edge Disposable High Profile Microtome Blades</td>
			<td>Electron Microscopy Sciences</td>
			<td>63068-HP</td>
			<td></td>
		</tr>
		<tr>
			<td>Transparent vacuum tubing</td>
			<td>Cole Palmer</td>
			<td>EW-06414-30</td>
			<td></td>
		</tr>
		<tr>
			<td>Ultragrade 19 vacuum pump oil</td>
			<td>Edwards</td>
			<td>H11025011</td>
			<td></td>
		</tr>
		<tr>
			<td>Variable voltage transformer</td>
			<td>Powerstat</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Water, suitable for HPLC</td>
			<td>Sigma Aldrich</td>
			<td>7732-18-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Wide-mouth dewar flask</td>
			<td>Sigma Aldrich</td>
			<td>Z120790</td>
			<td></td>
		</tr>
	</tbody>
</table>

investigation of microbial cooperation via imaging mass spectrometry analysis of bacterial colonies grown on agar and in tissue during infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical imaging. Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry represents a label-free, in situ imaging modality capable of generating spatial maps for a wide variety of metabolites. While thinly sectioned tissue samples are now routinely analyzed via this technology, imaging mass spectrometry analyses of non-traditional substrates, such as bacterial colonies commonly grown on agar in microbiology research, remain challenging due to the high water content and uneven topography of these samples. This paper demonstrates a sample preparation workflow to allow for imaging mass spectrometry analyses of these sample types. This process is exemplified using bacterial co-culture macrocolonies of two gastrointestinal pathogens: Clostridioides difficile and Enterococcus faecalis. Studying microbial interactions in this well-defined agar environment is also shown to complement tissue studies aimed at understanding microbial metabolic cooperation between these two pathogenic organisms in mouse models of infection. Imaging mass spectrometry analyses of the amino acid metabolites arginine and ornithine are presented as representative data. This method is broadly applicable to other analytes, microbial pathogens or diseases, and tissue types where a spatial measure of cellular or tissue biochemistry is desired.

We have performed metabolite MALDI imaging mass spectrometry of model bacterial colonies and mice co-colonized with E. faecalis and C. difficile to study the role of amino acids in microbe-microbe interactions. Bacterial macrocolonies grown on agar serve as a well-defined model to analyze distinct biochemical changes in bacterial biofilm formation. It is important to ensure a controlled drying process for bacterial culture macrocolonies grown on agar media to minimize deformations and cracking in the ag...

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Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

A novel sample preparation method is demonstrated for the analysis of agar-based, bacterial macrocolonies via matrix-assisted laser desorption/ionization imaging mass spectrometry.

Investigation of Microbial Cooperation via Imaging Mass Spectrometry Analysis of Bacterial Colonies Grown on Agar and in Tissue During Infection

Understanding the metabolic consequences of microbial interactions that occur during infection presents a unique challenge to the field of biomedical ...

Our protocol enables the visualization of spatial metabolism of microbial interactions that occur during infection. We have developed this imaging mass spectrometry workflow for bacterial co-cultures grown on agar and tissue models. Analyzing non-traditional substrates such as bacterial cultures grown on agar presents many challenges.Using our protocol, bacterial specimens can be appropriately dried to facilitate MALDI matrix application in subsequent imaging mass spectrometry analysis. This method is broadly applicable to a variety of metabolites, microbial pathogens or diseases, and sample types where a spatial measure of cellular or tissue biochemistry is desired, particularly when studying sample types that require dehydration prior to analysis. After preparing the bacterial culture macrocolonies, remove the agarose bacterial colonies mounted on ITO-coated microscope slides from the packaging material.Place the samples in a dry box with dessicant for 48 to 72 hours at room temperature. Visually inspect the colonies for deformations in the agar surface. Next, close the vacuum line to the sample chamber, and turn on the power switch to the rotary vane pump to allow the vacuum pump to warm up and achieve proper vacuum pressure.Turn on the variable voltage transformer to heat the wire filament wrapped around the chamber. Adjust the variable power supply until the internal temperature of the vacuum chamber reaches approximately 50 degrees Celsius. While the pump is warming up, place a slurry of dry ice and 100%ethanol into the cold trap condenser.Open the vacuum chamber using a wrench to loosen the 16-millimeter double cloth flange clamps. Insert the agarose samples into the chamber, and seal the chamber tightly with the 16-millimeter double cloth flange clamps. Then open the pump valve to evacuate the chamber, and allow the samples to dry for 60 to 120 minutes.When complete, slowly vent the vacuum chamber to ambient pressure by closing the valve on the rotary vane pump, and opening the vacuum flange to ambient air. Open the chamber as demonstrated previously, and remove the dried agarose sample from the chamber. Use 1, 5-Diaminonaphthalene MALDI matrix for its favorable desorption and ionization of amino acids and negative ion mode.Attach the sample to the sprayer tray, and load the prepared solutions into the sprayer line. Using the computer software, specify the necessary parameters for a uniform coating of the matrix compound. Monitor the spraying sequence until it is finished.Remove the sample from the sprayer tray, and store it in a desiccation cabinet until analysis. Insert the matrix-coated sample into the MALDI target plate microscope slide adapter. Inscribe at least three fiducial markers that encompass the sample area using permanent markers.Use a flatbed scanner to acquire an optical image of the microscope slide including the fiduciary markers. Now define a mass spectrometry instrument method as described in the manuscript. In negative ion mode, select the 100 dalton mass window from mass by charge 100 to 200 for gas phase signal enrichment using a continuous accumulation of selected ions approach which encompasses the mass by charge values of the metabolites of interest.Open the instrument's image acquisition software, and use the setup wizard to define the file name and location, the MS acquisition mode, regions of interest to be sampled, and the spatial resolution of the image. Teach the sample by using the fiducial markers from the optical image to synchronize with the instrument sample carrier. Then define the regions of interest on the optical image using the software's regions of interest selection tool.Once all the parameters have been defined, start the acquisition sequence to serially acquire a mass spectrum at each pixel across the defined region of interest. Following image acquisition, save the data with a MIS file extension which is a vendor-specific file format for imaging mass spectrometry platforms. Open the data file in flexImaging, or SCiLS software packages, or export to a non-proprietary data format such as mzML, and visualize using vendor neutral software.Using the reference software, define mass windows for peaks of interest to generate false color heat maps of ion distributions across the sampled regions. On the average mass spectrum display, zoom in to the peak of interest, and select an appropriate mass window that encompasses the area of the peak profile. Now, right-click on the highlighted mass window, and select Add Mass Filter.Label the selected mass by charge value using the tentative identification of the suspected metabolite as determined by the high-resolution accurate mass measurement. Select the intensity threshold to adjust the dynamic range of the analyte image displayed by the false color heat map. Further adjust the mass-filtering parameters so that the mass window encompasses the area of the peak profile, and then add the mass filter.Clean all cryosectioning equipment by rinsing it with 100%ethanol, and allow to dry before placing samples into the cryostat chamber. Prepare ITO-coated microscope slides using an ohmmeter to identify the side of the slide with the conductive coating. Using a diamond-tipped scribe, etch and label the ITO-coated side of the microscope slide.Then perform cryosectioning on a research cryomicrotome. Transfer the OCT-embedded mouse ceca tissues from a 80 degree Celsius freezer to the cryomicrotome chamber on dry ice. Inside the cryomicrotome chamber, mount the OCT-embedded tissue on a sample chuck using an additional OCT solution.After the OCT solution has solidified, fix the chuck to the specimen head and begin cryosectioning at 10 to 50 micrometers increments to reach the desired tissue depth or plane of the organ. Once an optimal cross-section of the tissue is reached, begin collecting sections at 12 micrometers thickness. Gently manipulate the slice using artist paintbrushes, and place it onto a Teflon-coated microscope slide.To pick up the tissue from the Teflon-coated slide, roll the ITO-coated microscope slide on top of the tissue section. Thaw-mount the tissue section to the microscope slide by pressing the palm to the back of the ITO-coated slide. Continue to thaw-mount until the tissue section transitions from translucent to an opaque or matte texture.For same day analysis, transfer the tissue-mounted microscope slides on dry ice to a dry box with dessicant for storage. Perform the MALDI matrix application and MALDI imaging mass spectrometry as demonstrated earlier. Analyze the ion images from multiple biological replicates, and compare the results for a significance via intensity box plot comparisons using the SCiLS statistical software.Negative ion mode MALDI analysis of bacterial culture samples allows for detecting dozens of metabolites. High-resolution accurate mass measurements allow for the tentative identification of ornithine and arginine. The measurement regions for imaging mass spectrometry analysis are shown.Imaging mass spectrometry of these samples allows for the spatial mapping of both ornithine and arginine amino acid metabolites which are found to be altered and differentially localized in the presence of Enterococcus faecalis. The tissue morphology of infected mouse ceca was visualized using hematoxylin and eosin staining. Optical images of mouse ceca tissues after applying a 1, 5-Diaminonaphthalene MALDI matrix layer are shown.MALDI ion images for ornithine and arginine display unique spatial distributions across the tissue samples. The most important consideration for this protocol is to maintain gentle drying and handling of bacterial culture specimens to minimize bubbling, cracking, and other deformation of the sample. Following imaging mass spectrometry, bright-field and fluorescence microscopy analyses of tissue samples can be performed to evaluate tissue morphology.This can also enable targeted validation of molecular pathways using antibody-based imaging methods.

investigation-microbial-cooperation-via-imaging-mass-spectrometry

Research

JoVE Journal

Biochemistry

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the gastrointestinal tract, as resident microbiota occurs and prevents colonisation by exogenous bacteria. Indeed, more than 600 species of bacteria are found in the oral cavity, and a single individual may carry around 100 different at any time. Oral bacteria possess the ability to adhere to the various niches in the oral ecosystem, thus becoming integrated within the resident microbial communities, and favouring growth and survival. However, the flow of bacteria into the gut during swallowing has been proposed to disturb the balance of the gut microbiota. In fact, oral administration of P. gingivalis shifted bacterial composition in the ileal microflora. We used a synthetic community as a simplified representation of the natural oral ecosystem, to elucidate the survival and viability of oral bacteria subjected to simulated gastrointestinal transit conditions. Fourteen species were selected, subjected to in vitro salivary, gastric, and intestinal digestion processes, and presented to a multicompartment cell model containing Caco-2 and HT29-MTX cells to simulate the gut mucosal epithelium. This model served to unravel the impact of swallowed bacteria on cells involved in the enterohepatic circulation. Using synthetic communities allows for controllability and reproducibility. Thus, this methodology can be adapted to assess pathogen viability and subsequent inflammation-associated changes, colonization capacity of probiotic mixtures, and ultimately, potential bacterial impact on the presystemic circulation.

Assessing the Viability of a Synthetic Bacterial Consortium on the In Vitro Gut Host-microbe Interface

Gut host-microbe interactions were assessed using a novel approach combining a synthetic oral community, in vitro gastrointestinal digestion, and a model of the small intestine epithelium. We present a method that can be adapted to evaluate cell invasion of pathogens and multi-species biofilms, or even to test probiotic formulations&#39; survivability.

The interplay between host and microbiota has been long recognized and extensively described. The mouth is similar to other sections of the ...

Proofreading and DNA Repair Assay Using Single Nucleotide Extension and MALDI-TOF Mass Spectrometry Analysis

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have developed a simple non-labeled and non-radio-isotopic method using a matrix-assisted laser desorption ionization with time-of-flight (MALDI-TOF) mass spectrometry (MS) analysis for a proofreading study. Here, a DNA polymerase [e.g., the Klenow fragment (KF) of Escherichia coli DNA polymerase I (pol I) in this study] in the presence of all four dideoxyribonucleotide triphosphates is used to process a mismatched primer-template duplex. The mismatched primer is then proofread/extended and subjected to MALDI-TOF MS. The products are distinguished by the mass change of the primer down to single nucleotide variations. Importantly, a proofreading can also be determined for internal single mismatches, albeit at different efficiencies. Mismatches located at 2-4-nucleotides (nt) from the 3' end were efficiently proofread by pol I, and a mismatch at 5 nt from the primer terminus showed only a partial correction. No proofreading occurred for internal mismatches located at 6 - 9 nt from the primer 3' end. This method can also be applied to DNA repair assays (e.g., assessing a base-lesion repair of substrates for the endo V repair pathway). Primers containing 3' penultimate deoxyinosine (dI) lesions could be corrected by pol I. Indeed, penultimate T-I, G-I, and A-I substrates had their last 2 dI-containing nucleotides excised by pol I before adding a correct ddN 5'-monophosphate (ddNMP) while penultimate C-I mismatches were tolerated by pol I, allowing the primer to be extended without repair, demonstrating the sensitivity and resolution of the MS assay to measure DNA repair.

A non-labeled, non-radio-isotopic method for DNA polymerase proofreading and a DNA repair assay was developed by using high-resolution MALDI-TOF mass spectrometry and a single nucleotide extension strategy. The assay proved to be very specific, simple, rapid, and easy to perform for proofreading and repair patches shorter than 9-nucleotides.

The maintenance of the genome and its faithful replication is paramount for conserving genetic information. To assess high fidelity replication, we have ...

Leaf Spray Mass Spectrometry: A Rapid Ambient Ionization Technique to Directly Assess Metabolites from Plant Tissues

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing plant metabolites because it provides molecular weights with high sensitivity and specificity. Leaf spray MS is an ambient ionization technique where plant tissue is used for direct chemical analysis via electrospray, eliminating chromatography from the process. This approach to sampling metabolites allows for a wide range of chemical classes to be detected simultaneously from intact plant tissues, minimizing the amount of sample preparation needed. When used with a high-resolution, accurate mass MS, leaf spray MS facilitates the rapid detection of metabolites of interest. It is also possible to collect tandem mass fragmentation data with this technique to facilitate a compound identification. The combination of accurate mass measurements and fragmentation is beneficial in confirming compound identities. The leaf spray MS technique requires only minor modifications to a nanospray ionization source and is a useful tool to further expand the capabilities of a mass spectrometer. Here, fresh leaf tissue from Sceletium tortuosum (Aizoaceae), a traditional medicinal plant from South Africa, is analyzed; numerous mesembrine alkaloids are detected with leaf spray MS.

Leaf spray mass spectrometry is a direct chemical analysis technique that minimizes the sample preparation and eliminates chromatography, allowing for the rapid detection of small molecules from plant tissues.

Plants produce thousands of small molecules that are diverse in their chemical properties. Mass spectrometry (MS) is a powerful technique for analyzing ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Untargeted Liquid Chromatography-Mass Spectrometry-Based Metabolomics Analysis of Wheat Grain

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management of product yield and quality. Metabolomics data provides a read-out of these interactions at a given moment in time and is informative of an organism&#39;s biochemical status. Further, individual metabolites or panels of metabolites can be used as precise biomarkers for yield and quality prediction and management. The plant metabolome is predicted to contain thousands of small molecules with varied physicochemical properties that provide an opportunity for a biochemical insight into physiological traits and biomarker discovery. To exploit this, a key aim for metabolomics researchers is to capture as much of the physicochemical diversity as possible within a single analysis. Here we present a liquid chromatography-mass spectrometry-based untargeted metabolomics method for the analysis of field-grown wheat grain. The method uses the liquid chromatograph quaternary solvent manager to introduce a third mobile phase and combines a traditional reversed-phase gradient with a lipid-amenable gradient. Grain preparation, metabolite extraction, instrumental analysis and data processing workflows are described in detail. Good mass accuracy and signal reproducibility were observed, and the method yielded approximately 500 biologically relevant features per ionization mode. Further, significantly different metabolite and lipid feature signals between wheat varieties were determined.

A method for the untargeted analysis of wheat grain metabolites and lipids is presented. The protocol includes an acetonitrile metabolite extraction method and reversed phase liquid chromatography-mass spectrometry methodology, with acquisition in positive and negative electrospray ionization modes.

Understanding the interactions between genes, the environment and management in agricultural practice could allow more accurate prediction and management ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.