We thank Heather Maughan and Linda M. Kalikin for careful editing of this manuscript. This work was supported by NIH NHLBI (grant 5R01HL136647-04).

1. Headspace Sorbent Pen (HSP) and sample analysis considerations
NOTE: The HSP containing the sorbent Tenax TA was selected to capture a broad range of volatiles. Tenax has a lower affinity for water compared to other sorbents, which enables it to trap more VOCs from higher-moisture samples. Tenax also has a low level of impurities and can be conditioned for re-use. Sorbent selection was also made in consideration with the column installed in the GC-MS (see the Table of Materials).
<ol>
	<li>Generate negative controls by extracting media and/or sample blanks with the same conditions used for sample extraction.</li>
	<li>Analyze a blank HSP (previously confirmed to be clean and free of significant background) on the GC-MS before analyzing extracted samples. Run blanks between sample types (e.g., three replicates of bacteria mono-culture, blank, three replicates of bacteria co-culture, blank, etc.).</li>
	<li>Limit use of fragrant personal care items or consumption of smelly foods prior to sample extraction and analysis. Ideally, prepare samples in a biosafety hood that has not been cleansed by alcohol or other volatile cleaners for at least 30 min. Turn on airflow in the biosafety hood for 30-60 min prior to sample preparation.</li>
	<li>Keep samples on ice to limit volatile release during sample preparation.</li>
</ol>
2. Mono- and co-culture preparation
<ol>
	<li>In the biosafety hood, inoculate cultures of A. baumannii, S. aureus, and P. aeruginosa in Todd Hewitt growth media. Incubate overnight at 37 &#176;C with 200 rpm agitation.</li>
	<li>After the overnight incubation, perform culture handling in the biosafety hood. Dilute each culture to optical density 0.05 at 500 nm.</li>
	<li>Mix co-cultures in equal parts, and pipette 200 &#181;L of control media, mono-, or co-culture into each well of a 96-well plate, and place in 37 &#176;C incubator for 24 h. Prepare a second plate for a 48-h incubation.</li>
	<li>At the end of the incubation period, prepare samples for extraction in section 4. Pipette liquid cultures into microcentrifuge tubes and store at -80 &#176;C. 
	NOTE: At this point, samples can be stored at -80 &#176;C to extract later if needed.</li>
</ol>
3. Stable isotope probing in biological samples preparation
NOTE: The feces and saliva samples were donated from anonymous donors with approval from the University of California Irvine Institutional Review Board (HS# 2017-3867). The sewage came from San Diego, CA. The sputum samples were collected from subjects with cystic fibrosis as part of a larger study approved by the University of Michigan Medical School Institutional Review Board (HUM00037056).
<ol>
	<li>Perform all biological sample preparations in the biosafety hood.
	<ol>
		<li>To prepare fecal samples, add 1 mL of deionized water to 100 mg of feces in a 1.5 mL microcentrifuge tube and vortex for 3 min. Place on ice when not in use.
		<ol>
			<li>To 15 &#181;L of fecal and water mixture, add 485 &#181;L of Brain Heart Infusion (BHI) medium with 20 mM 13C glucose, or BHI with 30% deuterium (D2O). Ensure that the final volume of the sample is 500 &#181;L. Prepare samples in technical triplicates.</li>
		</ol>
		</li>
		<li>To prepare sewage samples, add 500 &#181;L of sewage to 500 &#181;L of BHI with 20 mM 13C glucose or BHI with 30% D2O for a total volume of 1 mL. Prepare samples in triplicate. Place on ice when not in use.</li>
		<li>To prepare saliva samples, add 50 &#181;L of saliva to 500 &#181;L of BHI with 20 mM 13C glucose or BHI with 30% D2O for a total volume of 550 &#181;L. Prepare samples in triplicate. Place on ice when not in use.</li>
		<li>To prepare sputum samples to compare the volatiles present in the sample prior to and after culturing, perform a first extraction with 15 &#181;L of sputum. Prepare samples in triplicate. Place on ice when not in use. Proceed to section 4 for sample extraction, and extract for 18 h at 37 &#176;C with 200 rpm agitation.</li>
		<li>After the completion of the first extraction of the uncultured sputum samples, save the vials with sputum. Add 500 &#181;L of BHI with 20 mM 13C glucose to the vials with sputum from 3.5.1. Place on ice when not in use.</li>
	</ol>
	</li>
	<li>Proceed to section 4 for sample extraction.</li>
</ol>
4. Sample extraction
<ol>
	<li>Place empty volatile organic analysis (VOA) vials (20 mL) on the cold plate, and place the cold plate on ice in the biosafety hood.</li>
	<li>Turn on the 5600 sorbent pen extraction unit (SPEU), and adjust to the desired temperature as required for each method. 
	NOTE: For stable isotope probing experiments at 37 &#176;C, reaching the setpoint can take up to 15 min. For mono- and co-culture experiments at 70 &#176;C, reaching the setpoint can take up to 60 min.</li>
	<li>Collect clean HSPs that are equal to the number of samples prepared, including HSPs for media or sample controls.</li>
	<li>Label 20 mL VOA vials according to samples, replicates, and HSP IDs as needed. Use a marker that resists water in case condensation forms on the outside of the vial while on ice.</li>
	<li>Inside the biosafety hood, unscrew the white cap on the vial, quickly pipet sample into the vial, and assemble the black cap, lid liner, and HSP. 
	NOTE: Samples should not come into contact with the HSP, and sample volume will depend on sample type.</li>
	<li>Place the vial containing the sample and HSP back on the cold plate.</li>
	<li>Repeat steps 4.5 and 4.6 for each sample. Perform these steps per sample instead of all at once to prevent sample warming and thus, premature volatile release.</li>
	<li>Once all samples have been prepared in the glass vials, perform the following steps outside the biosafety hood on the bench. Turn on the vacuum pump, place the vials under vacuum to 30 mmHg, and remove the vacuum source. 
	NOTE: The vials do not need to be on the cold tray after vacuum application has been completed.</li>
	<li>Double-check the pressure after placing all samples under vacuum using the pressure gauge. If a vial has a leak, ensure that the cap is screwed on tightly, and that the white O-rings of the HSP and lid liners are properly in place. 
	NOTE: A compromised seal can result in decreased volatile detection compared to a vial under vacuum.</li>
	<li>Place vials in the SPEU for the optimized time and temperature with agitation at 200 rpm. Extract cultures for 1 h at 70 &#176;C. Extract stable isotope probing experiments with fecal, sewage, saliva, and sputum samples for 18 h at 37 &#176;C.</li>
	<li>Place the cold plate at -80 &#176;C for use after the extraction period is complete.</li>
	<li>When extraction is complete, place samples on the cold plate for 15 min to draw out water vapor from the HSP and vial headspace.</li>
	<li>Transfer the HSPs to their sleeves. 
	&#8203;NOTE: The experiment can be paused here for up to ~1 week at room temperature before losing the more highly volatile compounds from the HSPs.</li>
</ol>
5. Analyze samples on the gas chromatography - mass spectrometer (GC-MS)
<ol>
	<li>Use the following GC-MS (see the Table of Materials) settings: 35 &#176;C with a 5 min hold, 10 &#176;C/min ramp to 170 &#176;C, and a 15 &#176;C/min ramp to 230 &#176;C with a 20:1 split ratio and a total runtime of 38 min.</li>
	<li>Set the desorption method as follows: 2 min, 70 &#176;C preheat; 2 min 260 &#176;C desorption; 34 min, 260 &#176;C bakeout; and 2 min, 70 &#176;C post bake.</li>
	<li>Set up the sequence of samples, and start the run according to instrumentation.
	<ol>
		<li>To set up a sequence on the Entech Software, open the program. In the options to the right of the instrument dropdown menu, select 5800 | Sequence.</li>
		<li>Observe the sequence table in the Entech software similar to that in the GC-MS software. Name the Sample ID column according to Current date_vial number. Keep in mind that Name is analogous to Name in the GC-MS sequence table, and 5800 Method determines the rate of temperature ramp, holding times, etc. (opens a menu to select the method generated in step 5.2).</li>
		<li>Keep in mind that the Tray and Position columns determine where the Sample Preparation Rail (SPR) will go to pick up the HSPs.
		<ol>
			<li>Observe the two trays with 30 spots each to the immediate left, laid out as six columns with five spots each.&#160;The tray position that is left most and closest to the user (front) is position 1, while the rightmost, furthest away is position 30.</li>
			<li>Note that these trays are HSP A or B, where HSP B is the tray closer to the SPR (innermost tray), and directly behind HSP B is HSP Blank. Place the extracted samples into the trays, and select the spot on the sequence accordingly.</li>
		</ol>
		</li>
		<li>Save the sequence table, select Run on the left-hand side, then Start with blank in desorber if the blank HSP is in the desorber (denoted by a HSP marked by yellow label).</li>
	</ol>
	</li>
	<li>Note that HSPs will be handled by the SPR for each sample in the sequence. Let the SPR warm up, then a message will appear at the top of the screen to confirm if the blank is in the desorber. Click on Skip to confirm that the pen is there. Allow the SPR to run all samples automatically, and the sequence on the GC-MS side will automatically record the data in separate files.</li>
</ol>
6. Data analysis
<ol>
	<li>Quality-filter data on GC-MS software (Table of Materials).
	<ol>
		<li>Review each peak on the chromatogram, and annotate peaks that match the National Institute of Standards &#38; Technology (NIST) library (or with another available library).</li>
		<li>Add annotated chromatogram peaks to the processing method. Set the criteria for selecting peaks to include compounds with a greater than 75% probability, and ensure that the alignment of each identifying ion of the compound lies within the center of the peak.
		<ol>
			<li>To add a peak to the processing method, select Calibrate | Edit Compound | Name | insert compound under External Standard Compound. Add the name of the compound, retention time, Quant Signal Target Ion. Add the three largest peaks. To save, select ok | Method | Save.</li>
		</ol>
		</li>
		<li>Once the process method is set up, proceed to Quantitate | Calculate, and View | QEdit Quant Result.</li>
		<li>Inspect each compound to ensure that the peaks align with their expected retention times and are above background noise.</li>
		<li>Once QEdit has been completed, select Exit | Yes to save the QEdits and return to the main chromatogram. Export the area integrations by opening the file on the left-hand side. Select Quantitate | Generate Report.</li>
		<li>To export files for use in DExSI, select File | Export Data to AIA format | Create New Directory, and select a location for the file or Use Existing Directory.</li>
		<li>Observe a new window opening up to select files for export. Move the files to the right side of the window and click on Process. Wait for a few seconds to a few minutes depending on the number of files being converted.</li>
	</ol>
	</li>
	<li>Correct for isotope abundance in DExSI according to instructions for the DExSI software (https://github.com/DExSI/DExSI), and perform analysis with a favorite software or program (e.g., R).&#160;Scripts used to generate the figures are located at&#160;<a data-saferedirecturl="https://www.google.com/url?q=https://github.com/joannlp/VOC_SIP&amp;source=gmail&amp;ust=1646398895210000&amp;usg=AOvVaw18Jx-lKhdf3eNanCnF4OhQ" href="https://github.com/joannlp/VOC_SIP" target="_blank">https://github.com/joannlp/VOC_SIP</a>.</li>
</ol>

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V. L. V and S. J. B. D. were former employees of Entech Instruments Inc., and K. W. is a member of Entech&#39;s University Program. J. P., J. K., and C. I. R. have no conflicts of interest to declare.

To identify volatile production in in vitro cultures and human-associated samples, volatile analysis of mono- and co-cultures of P. aeruginosa, S. aureus, and A. baumanii and stable isotope probing of different biological samples were performed. In the analysis for the mono- and co-cultures, volatiles were detected by performing a short extraction for 1 h at 70 &#176;C. The volatile analysis of mono- and co-cultures allowed the survey of the compounds produced both by individual species and dur...

Volatile organic compounds (VOCs) have great promise for bacterial detection and identification because they are emitted from all organisms, and different microbes have unique VOC signatures. Volatile molecules have been utilized as a non-invasive measurement for detecting various respiratory infections including chronic obstructive pulmonary disease1, tuberculosis2 in urine3, and ventilator-associated pneumonia4, in addition to distinguishing subjects with cystic fibrosis (CF) from healthy control subjects5,6. Volatile signatures have even been used to distinguish specific pathogen infections in CF (Staphylococcus aureus7, Pseudomonas aeruginosa8,9, and S. aureus vs. P. aeruginosa10). However, with the complexity of such biological samples, it is often difficult to pinpoint the source of specific VOCs.
One strategy for disentangling the volatile profiles from multiple infecting microbes is to perform headspace analysis of microorganisms in both mono- and co-culture11. Headspace analysis examines the analytes emitted into the &#34;headspace&#34; above a sample rather than those embedded in the sample itself. Microbial metabolites have often been characterized in mono-cultures because of the difficulty in determining the origin of microbial metabolites in complex clinical samples. By profiling volatiles from bacterial mono-cultures, the types of volatiles a microbe produces in vitro may represent a baseline of its volatile repertoire. Combining bacterial cultures, e.g., creating co-cultures, and profiling the volatile molecules produced may reveal the interactions or cross-feeding between the bacteria12.
Another strategy for identifying the microbial origin of volatile molecules is to provide a nutrient source that is labeled with a stable isotope. Stable isotopes are naturally occurring, non-radioactive forms of atoms with a different number of neutrons. In a strategy that has been utilized since the early 1930s to trace active metabolism in animals13, the microorganism feeds off of the labeled nutrient source and incorporates the stable isotope into its metabolic pathways. More recently, a stable isotope in the form of heavy water (D2O) has been used to identify metabolically active S. aureus in a clinical CF sputum sample14. In another example, 13C-labeled glucose has been used to demonstrate the cross-feeding of metabolites between CF clinical isolates of P. aeruginosa and Rothia mucilaginosa12 .
With the advancement of mass spectrometry techniques, methods of detecting volatile cues have moved from qualitative observations to more quantitative measurements. By using gas chromatography mass spectrometry (GC-MS), processing of biological samples has become within reach for most laboratory or clinical settings. Many methods to survey volatile molecules have been used to profile samples such as food, bacterial cultures, and other biological samples, and air and water to detect contamination. However, several common methods of volatile sampling with high-throughput require solvent and are not performed with the advantages provided by vacuum extraction. In addition, larger volumes or quantities (greater than 0.5 mL) of sampled materials are often required for analysis15,16,17,18,19, although this is substrate-specific and requires optimization for each sample type and method.
Here, vacuum-assisted sorbent extraction (VASE) followed by thermal desorption on a GC-MS was employed to survey the volatile profiles of bacterial mono- and co-cultures and identify actively produced volatiles with stable isotope probing from human feces, saliva, sewage, and sputum samples (Figure 1). With limited sample quantities, VOCs were extracted from as little as 15 &#181;L of sputum. Isotope probing experiments with human samples required adding a stable isotope source, such as 13C glucose, and media to cultivate the growth of the microbial community. The active production of volatiles was identified as a heavier molecule by GC-MS. Extraction of volatile molecules under a static vacuum enabled the detection of volatile molecules with increased sensitivity20,21,22.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>13C glucose</td>
			<td>Sigma-Aldrich</td>
			<td>389374-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Stg Diaph Pump</td>
			<td>Entech Instruments</td>
			<td>01-10-20030</td>
			<td></td>
		</tr>
		<tr>
			<td>20 mL VOA vials</td>
			<td>Fisher Scientific</td>
			<td>5719110</td>
			<td></td>
		</tr>
		<tr>
			<td>24 mm Black Caps with hole, no septum</td>
			<td>Entech Instruments</td>
			<td>01-39-76044B</td>
			<td>holds lid liner in place on vial</td>
		</tr>
		<tr>
			<td>24 mm vial liner for sorbent pens</td>
			<td>Entech Instruments</td>
			<td>SP-L024S</td>
			<td>allows pens to make a vacuum seal at top of vial</td>
		</tr>
		<tr>
			<td>5600 Sorbent pen extraction unit (SPEU)</td>
			<td>Entech Instruments</td>
			<td>5600-SPES</td>
			<td>5600 Sorbent Pen Extraction Unit -120 VAC</td>
		</tr>
		<tr>
			<td>96-well assay plates</td>
			<td>Genesee</td>
			<td>25-224</td>
			<td></td>
		</tr>
		<tr>
			<td>Brain Heart Infusion (BHI) media</td>
			<td>Sigma-Aldrich</td>
			<td>53286-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>ChemStation Stofware</td>
			<td>Agilent</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>DB-624 column</td>
			<td>Agilent</td>
			<td>122-1364E</td>
			<td>60 m, 0.25 mm ID, 1.40 micron film thickness, in GC-MS</td>
		</tr>
		<tr>
			<td>Deuterium oxide</td>
			<td>Sigma-Aldrich</td>
			<td>151882-1L</td>
			<td></td>
		</tr>
		<tr>
			<td>Dexsi sofware</td>
			<td>Dexsi (open source)</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>GC-MS (7890A GC and 5975C inert XL MSD with Triple-Axis Detector)</td>
			<td>Agilent</td>
			<td></td>
			<td>7890A GC and 5975C inert XL MSD with triple-axis detector</td>
		</tr>
		<tr>
			<td>Headspace Bundle HS-B01, 120VA</td>
			<td>Entech Instruments</td>
			<td>SP-HS-B01</td>
			<td>Items for running headspace extraction included in bundle</td>
		</tr>
		<tr>
			<td>Headspace sorbent pen (HSP) - blank</td>
			<td>Entech Instruments</td>
			<td>SP-HS-0</td>
			<td></td>
		</tr>
		<tr>
			<td>Headspace sorbent pen (HSP) Tenax TA (35/60 Mesh)</td>
			<td>Entech Instruments</td>
			<td>SP-HS-T3560</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge tubes (2 mL)</td>
			<td>VWR</td>
			<td>53550-792</td>
			<td></td>
		</tr>
		<tr>
			<td>O-rings</td>
			<td>Entech Instruments</td>
			<td>SP-OR-L024</td>
			<td></td>
		</tr>
		<tr>
			<td>Sample Preparation Rail</td>
			<td>Entech Instruments</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Sorbent pen thermal conditioner</td>
			<td>Entech Instruments</td>
			<td>3801-SPTC</td>
			<td></td>
		</tr>
		<tr>
			<td>Todd Hewitt (TH) media</td>
			<td>Sigma</td>
			<td>T1438-500G</td>
			<td></td>
		</tr>
	</tbody>
</table>

capturing actively produced microbial volatile organic compounds from human-associated samples with vacuum-assisted sorbent extraction

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the host&#39;s microbial community. To disentangle the origin of microbial VOCs, volatile headspace analysis of bacterial mono- and co-cultures of Staphylococcus aureus, Pseudomonas aeruginosa, and Acinetobacter baumannii, and stable isotope probing in biological samples of feces, saliva, sewage, and sputum were performed. Mono- and co-cultures were used to identify volatile production from individual bacterial species or in combination with stable isotope probing to identify the active metabolism of microbes from the biological samples.
Vacuum-assisted sorbent extraction (VASE) was employed to extract the VOCs. VASE is an easy-to-use, commercialized, solvent-free headspace extraction method for semi-volatile and volatile compounds. The lack of solvents and the near-vacuum conditions used during extraction make developing a method relatively easy and fast when compared to other extraction options such as tert-butylation and solid phase microextraction. The workflow described here was used to identify specific volatile signatures from mono- and co-cultures. Furthermore, analysis of the stable isotope probing of human associated biological samples identified VOCs that were either commonly or uniquely produced. This paper presents the general workflow and experimental considerations of VASE in conjunction with stable isotope probing of live microbial cultures.

Mono- and co-cultures of S. aureus, P. aeruginosa, and A. baumannii 
The mono- and co-cultures consisted of the bacterial species S. aureus, P. aeruginosa, and A. baumannii. These are common opportunistic pathogens found in human wounds and chronic infections. To identify the volatile molecules present in the mono- and co-cultures, a short 1-h extraction was performed at ...

Watch this Scientific Journal Video about Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction at JoVE.com

Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing.

Volatile organic compounds (VOCs) from biological samples have unknown origins. VOCs may originate from the host or different organisms from within the ...

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Research

JoVE Journal

Biochemistry

3.9K Views.  University of California Irvine. This protocol describes the extraction of volatile organic compounds from a biological sample with the vacuum-assisted sorbent extraction method, gas chromatography coupled with mass spectrometry using the Entech Sample Preparation Rail, and data analysis. It also describes culture of biological samples and stable isotope probing. 

Video: Capturing Actively Produced Microbial Volatile Organic Compounds from Human-Associated Samples with Vacuum-Assisted Sorbent Extraction

Mimicking the Function of Signaling Proteins: Toward Artificial Signal Transduction Therapy

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins that interact with each other and activate one other with high specificity. Synthetic agents that mimic the function of these proteins might therefore be used to generate unnatural signal transduction steps and consequently, alter the cell's function. We present guidelines for designing 'chemical transducers' that can induce artificial communication between native proteins. In addition, we present detailed protocols for synthesizing and testing a specific 'transducer', which can induce communication between two unrelated proteins: platelet-derived growth-factor (PDGF) and glutathione-S-transferase (GST). The way by which this unnatural PDGF-GST communication could be used to control the cleavage of an anticancer prodrug is also presented, indicating the potential for using such systems in 'artificial signal transduction therapy'. This work is intended to facilitate developing additional 'transducers' of this class, which may be used to mediate intracellular protein-protein communication and consequently, to induce artificial cell signaling pathways.

We present guidelines for developing synthetic 'chemical transducers' that can induce communication between naturally unrelated proteins. In addition, detailed protocols are presented for synthesizing and testing a specific 'transducer' that enables a growth factor to activate a detoxifying enzyme and consequently, to regulate the cleavage of an anticancer prodrug.

Signal transduction pathways, which control the response of cells to various environmental signals, are mediated by the function of signaling proteins ...

Multimer-PAGE: A Method for Capturing and Resolving Protein Complexes in Biological Samples

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by networks of interacting protein complexes, which are often difficult to investigate because their binding is non-covalent and easily perturbed by purification techniques. This work describes a method of stabilizing and separating native protein complexes from unmodified tissue using two-dimensional polyacrylamide gel electrophoresis. Tissue lysate is loaded onto a non-denaturing blue-native polyacrylamide gel, then an electric current is applied until the protein migrates a short distance into the gel. The gel strip containing the migrated protein is then excised and incubated with the amine-reactive cross-linking reagent dithiobis(succinimidyl propionate), which covalently stabilizes protein complexes. The gel strip containing cross-linked complexes is then cast into a sodium dodecyl sulfate polyacrylamide gel, and the complexes are separated completely. The method relies on techniques and materials familiar to most molecular biologists, meaning it is inexpensive and easy to learn. While it is limited in its ability to adequately separate extremely large complexes, and has not been universally successful, the method was able to capture a wide variety of well-studied complexes, and is likely applicable to many systems of interest.

A method for stabilizing and separating native protein complexes from unmodified tissue lysate using an amine-reactive protein cross-linker coupled to a novel two-dimensional polyacrylamide gel electrophoresis (PAGE) system is presented.

There are many well-developed methods for purifying and studying single proteins and peptides. However, most cellular functions are carried out by ...

Optimized Protocol for the Extraction of Proteins from the Human Mitral Valve

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit the large-scale identification and quantification of the proteins present in complex biological systems.The knowledge gained from a proteomic approach can potentially lead to a better understanding of the pathogenic mechanisms underlying diseases, allowing for the identification of novel diagnostic and prognostic disease markers, and, hopefully, of therapeutic targets. However, the cardiac mitral valve represents a very challenging sample for proteomic analysis because of the low cellularity in proteoglycan and collagen-enriched extracellular matrix. This makes it challenging to extract proteins for a global proteomic analysis. This work describes a protocol that is compatible with subsequent protein analysis, such as quantitative proteomics and immunoblotting. This can allow for the correlation of data concerning protein expression with data on quantitative mRNA expression and non-quantitative immunohistochemical analysis. Indeed, these approaches, when performed together, will lead to a more comprehensive understanding of the molecular mechanisms underlying diseases, from mRNA to post-translational protein modification. Thus, this method can be relevant to researchers interested in the study of cardiac valve physiopathology.

The protein composition of the human mitral valve is still partially unknown, because its analysis is complicated by low cellularity and therefore by low protein biosynthesis. This work provides a protocol to efficiently extract protein for the analysis of the mitral valve proteome.

Analysis of the cellular proteome can help to elucidate the molecular mechanisms underlying diseases due to the development of technologies that permit ...

Capturing the Interaction Kinetics of an Ion Channel Protein with Small Molecules by the Bio-layer Interferometry Assay

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe the application of this novel label-free technique to study the interaction of human EAG1 (hEAG1) channel proteins with the small molecule PIP2. hEAG1 channel has been recognized as potential therapeutic target because of its aberrant overexpression in cancers and a few gain-of-function mutations involved in some types of neurological diseases. We purified&#160;hEAG1 channel proteins from a mammalian stable expression system and measured the interaction with PIP2 by BLI. The successful measurement of the kinetics of binding between hEAG1 protein and PIP2 demonstrates that the BLI assay is a potential high-throughput approach used for novel small-molecule ligand screening in ion channel pharmacology.

The protocol here describes the interactions of purified hEAG1 ion channel protein with the small molecule lipid ligand phosphatidylinositol 4, 5-bisphosphate (PIP2). The measurement demonstrates that BLI could be a potential method for novel small-molecule ion channel ligand screening.

The bio-layer interferometry (BLI) assay is a valuable tool for measuring protein-protein and protein-small molecule interactions. Here, we first describe ...

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 ...

Profiling Volatile Compounds in Blackcurrant Fruit using Headspace Solid-Phase Microextraction Coupled to Gas Chromatography-Mass Spectrometry

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars with enhanced organoleptic characteristics and thus, to increase consumer acceptance. High-throughput metabolomic platforms have been recently developed to quantify a wide range of metabolites in different plant tissues, including key compounds responsible for fruit taste and aroma quality (volatilomics). A method using headspace solid-phase microextraction (HS-SPME) coupled with gas chromatography-mass spectrometry (GC-MS) is described here for the identification and quantification of VOCs emitted by ripe blackcurrant fruits, a berry highly appreciated for its flavor and health benefits.
Ripe fruits of blackcurrant plants (Ribes nigrum) were harvested and directly frozen in liquid nitrogen. After tissue homogenization to produce a fine powder, samples were thawed and immediately mixed with sodium chloride solution. Following centrifugation, the supernatant was transferred into a headspace glass vial containing sodium chloride. VOCs were then extracted using a solid-phase microextraction (SPME) fiber and a gas chromatograph coupled to an ion trap mass spectrometer. Volatile quantification was performed on the resulting ion chromatograms by integrating peak area, using a specific m/z ion for each VOC. Correct VOC annotation was confirmed by comparing retention times and mass spectra of pure commercial standards run under the same conditions as the samples. More than 60 VOCs were identified in ripe blackcurrant fruits grown in contrasting European locations. Among the identified VOCs, key aroma compounds, such as terpenoids and C6 volatiles, can be used as biomarkers for blackcurrant fruit quality. In addition, advantages and disadvantages of the method are discussed, including prospective improvements. Furthermore, the use of controls for batch correction and minimization of drift intensity have been emphasized.

A headspace solid-phase microextraction-gas-chromatography platform is described here for fast, reliable, and semi-automated volatile identification and quantification in ripe blackcurrant fruits. This technique can be used to increase knowledge about fruit aroma and to select cultivars with enhanced flavor for the purpose of breeding.

There is an increasing interest in measuring volatile organic compounds (VOCs) emitted by ripe fruits for the purpose of breeding varieties or cultivars ...

Absolute Quantitation of Inositol Pyrophosphates by Capillary Electrophoresis Electrospray Ionization Mass Spectrometry

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules feature the presence of at least one energetic pyrophosphate moiety on the myo-inositol ring. They exist ubiquitously in eukaryotes and operate as metabolic messengers surveying phosphate homeostasis, insulin sensitivity, and cellular energy charge. Owing to the absence of a chromophore in these metabolites, a very high charge density, and low abundance, their analysis requires radioactive tracer, and thus it is convoluted and expensive. Here, the study presents a detailed protocol to perform absolute and high throughput quantitation of inositol pyrophosphates from mammalian cells by capillary electrophoresis electrospray ionization mass spectrometry (CE-ESI-MS). This method enables the sensitive profiling of all biologically relevant PP-InsPs species in mammalian cells, enabling baseline separation of regioisomers. Absolute cellular concentrations of PP-InsPs, including minor isomers, and monitoring of their temporal changes in HCT116 cells under several experimental conditions are presented.

A procedure for capillary electrophoresis electrospray ionization mass spectrometry for the absolute quantitation of inositol pyrophosphates from mammalian cell extracts is described.

Inositol pyrophosphates (PP-InsPs) are an important group of intracellular signaling molecules. Derived from inositol phosphates (InsPs), these molecules ...

Reconstitution of Msp1 Extraction Activity with Fully Purified Components

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function depends on a robust quality control system to maintain protein homeostasis (proteostasis). Declines in mitochondrial proteostasis have been linked to cancer, aging, neurodegeneration, and many other diseases. Msp1 is a AAA+ ATPase anchored in the outer mitochondrial membrane that maintains proteostasis by removing mislocalized tail-anchored proteins. Using purified components reconstituted into proteoliposomes, we have shown that Msp1 is necessary and sufficient to extract a model tail-anchored protein from a lipid bilayer. Our simplified reconstituted system overcomes several of the technical barriers that have hindered detailed study of membrane protein extraction. Here, we provide detailed methods for the generation of liposomes, membrane protein reconstitution, and the Msp1 extraction assay.

Here, we present a detailed protocol for reconstitution of Msp1 extraction activity with fully purified components in defined proteoliposomes.

As the center for oxidative phosphorylation and apoptotic regulation, mitochondria play a vital role in human health. Proper mitochondrial function ...

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center of attention, specifically acidic cannabinoids. The focus is turning toward acidic cannabinoids due to their lack of psychotropic activity. Cannabis plants produce acidic cannabinoids with hemp plants producing low levels of psychotropic cannabinoids. As such, utilization of hemp for acidic cannabinoid extraction would eliminate the need for decarboxylation prior to extraction as a source for the cannabinoids. The use of solvent-based extraction is ideal for obtaining acidic cannabinoids as their solubility in solvents such as supercritical CO2 is limited due to the high pressure and temperature required to reach their solubility constants. An alternative method designed to increase solubility is ultrasonic-assisted extraction. In this protocol, the impact of solvent polarity (acetonitrile 0.46, ethanol 0.65, methanol 0.76, and water 1.00) and concentration (20%, 50%, 70%, 90%, and 100%) on ultrasonic-assisted extraction efficiency has been examined. Results show that water was the least effective and acetonitrile was the most effective solvent examined. Ethanol was further examined since it has the lowest toxicity and is generally regarded as safe (GRAS). Surprisingly, 50% ethanol in water is the most effective ethanol concentration for extracting the highest amount of cannabinoids from hemp. The increase in cannabidiolic acid concentration was 28% when compared to 100% ethanol, and 23% when compared to 100% acetonitrile. While it was determined that 50% ethanol is the most effective concentration for our application, the method has also been demonstrated to be effective with alternative solvents. Consequently, the proposed method is deemed effective and rapid for extracting acidic cannabinoids.

Ultrasonic-Assisted Extraction of Cannabidiolic Acid from Cannabis Biomass

Ultrasonic-assisted extraction (UAE) increases extraction efficiency of solvents and when applied to Cannabis spp. biomass it reduces the time required for extraction. This decreases the cost and potential cannabinoid loss due to degradation. Additionally, UAE is considered a green method due to low solvent use.

Industrial hemp (Cannabis spp.) has many compounds of interest with potential medical benefits. Of these compounds, cannabinoids have come to the center ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.