This work is funded by NIH grant P20 RR021905-01, CF RPD grant STANTO07R0, and NASA grant NNH09ZNE002C.

<img src="/files/ftp_upload/2664/2664fig1.jpg" alt="Figure 1"/> 
Figure 1. Schematic for SESI-MS analysis of bacterial volatiles. The headspace of the bacterial culture is displaced by CO2 (1) into the SESI reaction chamber (2). As the volatiles traverse the SESI reaction chamber they pass through the electrospray cloud and become ionized (3). Once ionized, the volatiles are pulled into the mass spectrometer for analysis (4). Excess carrier gas and unreacted bacterial volatiles are passed through a 0.22 &mu;m filter (5), as an additional measure of protection, and vented to a chemical hood. Inset: The SESI electrospray needle is a silica capillary (40 &mu;m ID) with a sharpened needle tip.
As a demonstration of the use of SESI-MS for the characterization of bacterial volatiles, E. coli K12 and P. aeruginosa PAO1 are cultured aerobically for 24 h in 50 mL LB-Lennox at 37 &deg;C and the SESI-MS spectra of the headspace volatiles are collected in 2 minutes. Carbon dioxide (99.99 %) at a flow rate of 2 L/min is used as the carrier gas for volatile delivery to the reaction chamber. The SESI reaction chamber was custom built and fitted to an API-3000 (SCIEX), replacing the original electrospray ion source. The spectra are collected in positive ion mode using 0.1 % formic acid, 5.0% methanol, and 94.9 % water (v/v) as the electrospray solution, delivered at 5 nL/s through a non-conductive silica capillary with a sharpened needle tip (40 &mu;m ID). The applied voltage is 2.5 kV. Analyst 1.4.2 software (Applied Biosystems) is used for data collection with the following parameters: 20 &#x2013; 500 Da, MCA mode, 40 scans, 3 s/scan, and 2 min total analysis time.
1. Culturing system
<ol>
 <li>Choose the appropriate vessel for growing your cultures, considering the growth requirements of the species in your experiment (e.g., aeration, light, 
 temperature, etc.), as well as the efficient delivery of volatiles to the mass spectrometer. The culture bottles we choose to use are standard 100 mL Pyrex 
 media bottles fitted with threaded caps that have at least two luer ports. An inlet line is inserted through one luer port for carrier gas delivery to the 
 sample bottle and an outlet line is inserted through another port for VOC delivery to the instrument (Figure 1). Any additional ports are plugged.</li>
 <li>Prior to culturing the samples, pressurize the vessels and submerge in water to check for leaks. Gas leaks are a primary cause of atypical 
 results in the form of weak or absent volatile ion signals.</li>
</ol>
2. Biological experiment: set-up and safety considerations
<ol>
 <li>Grow your cultures in the conditions appropriate to your hypothesis. It is recommended that at least two biological replicates, each with two technical replicates, are used for each variable.</li>
 <li>Prepare a blank for every culture condition (medium, antibiotics, etc.) and incubate the blank under the same conditions as your samples.</li>
 <li>Employ safety precautions that are appropriate for the biological agents you are using, taking into consideration the biosafety level(s) of the species.</li>
 <li>To prevent contamination of your instrument and the gas transfer lines with viable biological agents, install filters of the appropriate pore size into the carrier gas line. The filters will not interfere with the transfer of volatiles to the SESI reaction chamber, but may slightly impact the efficiency of aerosol transfer.6</li>
 <li>Use secondary containment or a biosafety cabinet when attaching the gas transfer cap to your culture bottle to assure the proper containment in the case of spilling biological agents.</li>
 <li>Initiate and terminate carrier gas flow to your sample bottle in a way that will not build pressure inside the bottle.</li>
</ol>
3. Instrument optimization
NOTE: SESI-MS is specifically designed to sample volatiles, so limit the use of fragrant personal care items (e.g., colognes, mouthwash, 
lotions, fabric softener), gum, cigarettes, etc. before using the instrument. Tightly cap all volatile chemicals in the lab, and control air drafts as much as 
possible during testing.
The following instrumental parameters, which all affect signal intensity and stability, will need to be optimized for your instrument and experiment.
<ol>
 <li>Electrospray solution and flow rate: Choose the appropriate electrospray solution for the class of molecules you wish to target, taking into account the operating instrument polarity (positive or negative-ion mode) and molecular character of the target compounds. In this experiment the electrospray solution is 0.1 % formic acid, 5.0 % methanol, 94.9 % water (v/v), which enhances the signal intensity of less polar molecules while providing good signal stability. The solution is delivered at a flow rate of 5 nL/s.</li>
 <li>Carrier gas flow rate: The carrier gas flow rate can affect the electrospray stability and the signal intensity. CO2 (&ge; 99.99%) at a flow rate of 2 L/min is used here. </li>
 <li>Needle shape and position: The needle tip shape and position strongly affect the signal intensity and stability. When installing a new needle, the position of the needle must be optimized to create a balance between low background, high analyte signal intensity, and signal stability. In order to reproduce SESI spectra after a needle change, it is necessary to periodically collect spectra as you adjust the needle position until you are able to match the observed spectrum to your records. The distance from electrospray needle tip to mass spec orifice will be 1 &#x2013; 5 mm.</li>
 <li>Applied voltage: The voltage that is applied to the system affects the signal ion intensity and the stability of the electrospray Taylor cone. 
 Additionally, the optimum voltage is dependent upon your electrospray solution and the needle tip shape. 
 At the start of your series of experiments, determine the voltage that yields the optimum spectrum and signal stability for your system, and then use this voltage for 
 all subsequent experiments. For our system, applied voltages of 2.0 - 5.0 kV provide optimal signal intensity and electrospray stability. For this experiment 2.5 kV is used.</li>
</ol>
4. Turning on and tuning the SESI-MS for analysis
<ol>
 <li>Begin by ensuring that the voltage supply is turned off and that the system is discharged of electricity. Do this by 1) ensuring the indicator lights on the voltage supply are off, 2) ensuring the voltage on the multimeter is zero, and 3) grounding the electrical leads.</li>
 <li>Install the appropriate electrospray solution for your experiment. </li>
 <li>Turn on the carrier gas and set the flow to the rate appropriate for your experiment.</li>
 <li>Apply pressure to the electrospray reservoir to initiate the delivery of the electrospray solution to the reaction chamber.</li>
 <li>Turn on the voltage supply and adjust the voltage to an appropriate value for your experiments.</li>
</ol>
NOTE: At this point the metal surfaces of the ionization source are capable of delivering a dangerous shock. Exercise great caution when working around the instrument once the voltage supply has been turned on.
<ol start="6">
 <li>Set up a tuning method for monitoring the SESI-MS spectrum while making fine-tuned adjustments to the applied voltage. Use the acquisition parameters that you have optimized for your system and your experiment. Clear the Multiple Channel Acquisition (MCA) check box (if applicable) so that every scan produces an independent spectrum, set the acquisition time to 10 &#x2013; 15 min, and start the acquisition. Spectra of the carrier gas background should now be observed.</li>
 <li>Make fine-tuned adjustments to the applied voltage to obtain a stable total ion chromatogram (TIC) and reproducible scans that match the &nbsp; CO2 scans for your previous experiments. Once the voltage adjustments have been made, continue to collect spectra and a TIC for five minutes to ensure the instrument is stabilized.</li>
 <li>Once instrumental stability is ensured, set up the acquisition method as appropriate for your samples, adjusting the acquisition time, data range, and MCA selection as needed. Collect a carrier gas background spectrum for your records.</li>
</ol>
5. Obtaining a volatile fingerprint of your bacterial culture
<ol>
 <li>To collect a blank spectrum, direct the carrier gas flow through the bypass lines, and then attach the blank sample (valves closed) to the gas transfer lines of the instrument.</li>
 <li>Open the valves to the sample bottle, and close the valves of the bypass lines.</li>
 <li>Allow the system to equilibrate for 30 seconds, during which time the humidity in the reaction chamber is stabilizing. This period of equilibration is essential for obtaining reproducible spectra. To ensure that the system is equilibrated, you may wish to monitor the TIC, which will change during the equilibration period, and stabilize thereafter.</li>
 <li>Once the system is equilibrated, initiate spectrum collection.</li>
 <li>After the spectrum is collected, remove the sample bottle by first opening the carrier gas bypass lines, then closing the sample valves, and lastly removing the sample bottle. Flush the system with the carrier gas for 2 &#x2013; 4 min, removing the moisture and adsorbed volatiles from the transfer lines, preventing sample-to-sample carryover.</li>
 <li>Repeat steps 5.2 - 5.5 for each bacterial sample, intermittently collecting additional blank spectra to ensure thorough blank subtraction. Incomplete blank subtraction will lead to the appearance of chemical background peaks in your processed spectrum that are common to atmospheric pressure ionization techniques, (e.g., phthalates, silicones, etc.).9</li>
 <li>When collecting your spectra, ensure that the ion signals are not exceeding the linear detection limits of your instrument, as determined by the TIC and the maximum intensity of individual peaks. Ions exceeding the upper limitations of your instrument&#x2019;s detector can generate artifact peaks that are not representative of your sample.</li>
</ol>
6. Representative Results
As an example of the SESI-MS spectra that can be obtained for bacterial volatiles, the positive ion mode volatile fingerprints for E. coli and P. aeruginosa grown aerobically in LB-Lennox for 24 h at 37 &deg;C are shown (Fig. 2). The E. coli volatile spectrum is dominated by indole at m/z = 118, which gives E. coli cultures their characteristic odor, whereas the spectrum of P. aeruginosa contains a larger variety of protonatable peaks.
Please note that the relative intensities of the peaks in the volatile spectrum are dependent upon the instrumental parameters described in Section 3. These parameters must be tightly controlled from experiment to experiment in order to obtain reproducible spectra.
<img src="/files/ftp_upload/2664/2664fig2.jpg" alt="Figure 2" /> 
Figure 2. Blank-subtracted positive ion mode SESI-MS spectra (20 &#x2013; 150 m/z) of E. coli K12 and P. aeruginosa PAO1 volatiles after 24 h aerobic growth in LB-Lennox at 37 &deg;C. For more details about the peaks observed in the SESI spectra, please refer to Zhu, et al. 8.

<ol>
	<li>Fuerstenau, S., Kiselev, P., Fenn, J. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=ESI-MS+in+the+analysis+of+trace+species+in+gases.">ESI-MS in the analysis of trace species in gases.</a> , (1999).</li><li>Wu, C., Siems, W. F., Hill, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+electrospray+ionization+ion+mobility+spectrometry/mass+spectrometry+of+illicit+drugs.">Secondary electrospray ionization ion mobility spectrometry/mass spectrometry of illicit drugs.</a> Anal. Chem. 72, 396-403 (2000).</li><li>Tam, M., Hill, H. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+electrospray+ionization-ion+mobility+spectrometry+for+explosive+vapor+detection.">Secondary electrospray ionization-ion mobility spectrometry for explosive vapor detection.</a> Anal. Chem. 76, 2741-2747 (2004).</li><li>Martinez-Lozano, P., Rus, J., de la Mora, G. F., Hernandez, M., de la Mora, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Secondary+electrospray+ionization+(SESI)+of+ambient+vapors+for+explosive+detection+at+concentrations+below+parts+per+trillion.">Secondary electrospray ionization (SESI) of ambient vapors for explosive detection at concentrations below parts per trillion.</a> J. Am. Soc. Mass Spectrom. 20, 287-294 (2009).</li><li>Martinez-Lozano, P., de la Mora, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-line+detection+of+human+skin+vapors.">On-line detection of human skin vapors.</a> J. Am. Soc. Mass Spectrom. 20, 1060-1063 (2009).</li><li>Martinez-Lozano, P., de la Mora, J. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrospray+ionization+of+volatiles+in+breath.">Electrospray ionization of volatiles in breath.</a> Int. J. Mass Spectrom. 265, 68-72 (2007).</li><li>Martinez-Lozano, P., de la Mora, J. F., F, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+analysis+of+fatty+acid+vapors+in+breath+by+electrospray+ionization+and+atmospheric+pressure+ionization-mass+spectrometry.">Direct analysis of fatty acid vapors in breath by electrospray ionization and atmospheric pressure ionization-mass spectrometry.</a> Anal. Chem. 80, 8210-8215 (2008).</li><li>Guo, X. H., Bruins, A. P., Covey, T. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+typical+chemical+background+interferences+in+atmospheric+pressure+ionization+liquid+chromatography-mass+spectrometry.">Characterization of typical chemical background interferences in atmospheric pressure ionization liquid chromatography-mass spectrometry.</a> Rapid Commun. Mass Spectrom. 20, 3145-3150 (2006).</li><li>Rudzinski, C. M., Herzig-Marx, R., Lin, J., Szpiro, A., Johnson, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Pathogen detection using headspace analysis. , 16-16 (2004).</li><li>Lechner, M., Fille, M., Hausdorfer, J., Dierich, M., Rieder, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Diagnosis+of+bacteria+in+vitro+by+mass+spectrometric+fingerprinting:+A+pilot+study.">Diagnosis of bacteria in vitro by mass spectrometric fingerprinting: A pilot study.</a> Curr. Microbiol. 51, 267-269 (2005).</li><li>Schulz, S., Dickschat, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+volatiles:+The+smell+of+small+organisms.">Bacterial volatiles: The smell of small organisms.</a> Nat. Prod. Rep. 24, 814-842 (2007).</li><li>Ligor, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analytical+methods+for+breath+investigation.">Analytical methods for breath investigation.</a> Crit. Rev. Anal. Chem. 39, 2-12 (2009).</li><li>Cao, W. Q., Duan, Y. X. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breath+analysis:+Potential+for+clinical+diagnosis+and+exposure+assessment.">Breath analysis: Potential for clinical diagnosis and exposure assessment.</a> Clin. Chem. 52, 800-811 (2006).</li><li>Law, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+characterization+of+complex+viscous+liquids+at+the+molecular+level.">Rapid characterization of complex viscous liquids at the molecular level.</a> Angew. Chem. Int. Ed. 48, 8277-8280 (2009).</li><li>Wu, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sampling+analytes+from+cheese+products+for+fast+detection+using+neutral+desorption+extractive+electrospray+ionization+mass+spectrometry.">Sampling analytes from cheese products for fast detection using neutral desorption extractive electrospray ionization mass spectrometry.</a> Anal. Bioanal. Chem. 397, 1549-1556 (2010).</li><li>Fleming-Jones, M. E., Smith, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Volatile+organic+compounds+in+foods:+A+five+year+study.">Volatile organic compounds in foods: A five year study.</a> J. Agric. Food Chem. 51, 8120-8127 (2003).</li><li>Calkins, C. R., Hodgen, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+fresh+look+at+meat+flavor.">A fresh look at meat flavor.</a> Meat Sci. 77, 63-80 (2007).</li></ol>

No conflicts of interest declared.

Bacteria produce different combinations of volatiles, which can be utilized for bacterial identification10-12 and the assessment of metabolic 
status. The SESI-MS method described here provides a means of rapidly characterizing bacterial volatiles (in two minutes or less) without any sample preparation, 
generating a bacterial "fingerprint" for the identification of the species.8 In the past several decades other atmospheric pressure ionization MS techniques have 
been applied to the characterization of volatile compounds, including selective ion flow tube (SIFT) and proton transfer reaction (PTR) mass spectrometry. 
The distinctive advantage that SESI provides over these other ionization methods is that it is possible to fragment specific peaks (provided the appropriate 
type of mass spectrometer has been adapted for SESI), which is an important tool for compound identification. We did not address peak fragmentation in the 
protocol listed above, but for examples of how fragmentation information can be used in the characterization of bacterial volatiles, please refer to Zhu, et al.8
SESI-MS has direct application to the in situ detection of bacterial lung infections via breath analysis, but can also
be applied to any setting in which volatile sampling is possible. For instance, the analyses of volatiles in urine, blood, and breath, which are 
relevant to the diagnosis of metabolic disorders, gastroenteric disease, cancer, and environmental exposure, are well suited to SESI-MS.13,14
SESI-MS also has a wide range of non-clinical VOC fingerprinting applications, including rapid analysis of foods for the characteristic 
volatiles associated with ripening, aging, or spoiling.15-18

<table border="1">
	<tbody>
		<tr>
			<td>Material Name</td>
			<td>Type</td>
			<td>Company</td>
			<td>Catalogue #</td>
			<td>Comment</td>
		</tr>
		<tr>
			<td>API-3000 Triple Quadrupole</td>
			<td>Instrument</td>
			<td>SCIEX</td>
			<td>&nbsp;</td>
			<td>Purchased with Analyst 1.4.2 (Applied Biosystems)</td>
		</tr>
		<tr>
			<td>SESI Ion Source</td>
			<td>Instrument</td>
			<td>&nbsp;</td>
			<td>&nbsp;</td>
			<td>Custom-made; See reference 6</td>
		</tr>
		<tr>
			<td>Gas flow meter</td>
			<td>Equipment</td>
			<td>Cole-Parmer</td>
			<td>EW-03217-74</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Carbon dioxide</td>
			<td>Equipment</td>
			<td>Airgas</td>
			<td>CD I300</td>
			<td>&ge; 99.99% pure</td>
		</tr>
		<tr>
			<td>Nitrogen</td>
			<td>Equipment</td>
			<td>Airgas</td>
			<td>NI UHP300</td>
			<td>Ultra high purity</td>
		</tr>
		<tr>
			<td>100 mL glass media bottles</td>
			<td>Equipment</td>
			<td>VWR</td>
			<td>89012-114</td>
			<td>GL45 screw threads</td>
		</tr>
		<tr>
			<td>Bottle caps with luer ports</td>
			<td>Equipment</td>
			<td>Bio Chem Fluidics</td>
			<td>00945T-3</td>
			<td>Cap assembly</td>
		</tr>
		<tr>
			<td>Luer port plugs</td>
			<td>Equipment</td>
			<td>Bio Chem Fluidics</td>
			<td>009LP</td>
			<td>Cap assembly</td>
		</tr>
		<tr>
			<td>Tubing 1/4" (OD) x 1/8" (ID)</td>
			<td>Equipment</td>
			<td>Cole-Parmer</td>
			<td>EW-95875-02</td>
			<td>Cap assembly & gas transfer lines</td>
		</tr>
		<tr>
			<td>Tubing 1/8" (OD) x 1/16" (ID)</td>
			<td>Equipment</td>
			<td>Cole-Parmer</td>
			<td>EW-06605-27</td>
			<td>Cap assembly</td>
		</tr>
		<tr>
			<td>Two-way valves</td>
			<td>Equipment</td>
			<td>Cole-Parmer</td>
			<td>07391-04</td>
			<td>Cap assembly</td>
		</tr>
		<tr>
			<td>Filter, Grade AAQ</td>
			<td>Equipment</td>
			<td>Balston Filters</td>
			<td>9922-05</td>
			<td>&nbsp;</td>
		</tr>
		<tr>
			<td>Formic acid, LC/MS grade</td>
			<td>Reagent</td>
			<td>Fisher</td>
			<td>A117-05AMP</td>
			<td>Electrospray solution</td>
		</tr>
		<tr>
			<td>Methanol, LC/MS grade</td>
			<td>Reagent</td>
			<td>Fisher</td>
			<td>A456-500</td>
			<td>Electrospray solution</td>
		</tr>
		<tr>
			<td>Water, LC/MS grade</td>
			<td>Reagent</td>
			<td>Fisher</td>
			<td>W6-500</td>
			<td>Electrospray solution</td>
		</tr>
	</tbody>
</table>

characterizing bacterial volatiles using secondary electrospray ionization mass spectrometry (sesi-ms)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for sample pretreatment. The method was first described by Fenn and colleagues1 and has been applied to the detection of illicit drugs2 and explosives3-4, the characterization of skin volatiles5, and the analysis of breath6-7.
SESI ionization occurs by proton transfer reactions between the electrospray solution and the volatile analyte, and is therefore suitable for the analysis of hetero-organic molecules, just as in traditional electrospray ionization (ESI). However, unlike standard ESI, the proton transfer process of SESI occurs in the vapor phase rather than in solution (Fig. 1), and therefore SESI is best suited for detecting organic volatiles and aerosols.
We are expanding the use of SESI-MS to the detection of bacterial volatiles as a method for bacterial identification and characterization8. We have demonstrated that SESI-MS volatile fingerprinting, combined with a statistical analysis method, can be used to differentiate bacterial genera, species, and mixed cultures in a variety of growth media.8 Here we provide the steps for obtaining bacterial volatile fingerprints using SESI-MS, including the instrumental parameters that should be optimized to ensure robust bacterial identification and characterization.

Watch this Scientific Journal Video about Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS at JoVE.com

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS)

Secondary electrospray ionization mass spectrometry (SESI-MS) is a method developed for the rapid detection of volatile compounds, without the need for ...

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17.8K Views.  University of Vermont. Secondary electrospray ionization mass spectrometry (SESI-MS) enables the detection of volatile organic compounds (VOCs) without the need for any sample pretreatment. This protocol provides instructions for the rapid (within minutes) characterization of bacterial VOCs using SESI-MS.

Video: Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry SESI-MS

Cellular Lipid Extraction for Targeted Stable Isotope Dilution Liquid Chromatography-Mass Spectrometry Analysis

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including numerous stereoisomers1,2. These metabolites can be formed from free or esterified fatty acids. Many of these oxidized metabolites have biological activity and have been implicated in various diseases including cardiovascular and neurodegenerative diseases, asthma, and cancer3-7. Oxidized bioactive lipids can be formed enzymatically or by reactive oxygen species (ROS). Enzymes that metabolize fatty acids include cyclooxygenase (COX), lipoxygenase (LO), and cytochromes P450 (CYPs)1,8. Enzymatic metabolism results in enantioselective formation whereas ROS oxidation results in the racemic formation of products.
While this protocol focuses primarily on the analysis of AA- and some LA-derived bioactive metabolites; it could be easily applied to metabolites of other fatty acids. Bioactive lipids are extracted from cell lysate or media using liquid-liquid (l-l) extraction. At the beginning of the l-l extraction process, stable isotope internal standards are added to account for errors during sample preparation. Stable isotope dilution (SID) also accounts for any differences, such as ion suppression, that metabolites may experience during the mass spectrometry (MS) analysis9. After the extraction, derivatization with an electron capture (EC) reagent, pentafluorylbenzyl bromide (PFB) is employed to increase detection sensitivity10,11. Multiple reaction monitoring (MRM) is used to increase the selectivity of the MS analysis. Before MS analysis, lipids are separated using chiral normal phase high performance liquid chromatography (HPLC). The HPLC conditions are optimized to separate the enantiomers and various stereoisomers of the monitored lipids12. This specific LC-MS method monitors prostaglandins (PGs), isoprostanes (isoPs), hydroxyeicosatetraenoic acids (HETEs), hydroxyoctadecadienoic acids (HODEs), oxoeicosatetraenoic acids (oxoETEs) and oxooctadecadienoic acids (oxoODEs); however, the HPLC and MS parameters can be optimized to include any fatty acid metabolites13.
Most of the currently available bioanalytical methods do not take into account the separate quantification of enantiomers. This is extremely important when trying to deduce whether or not the metabolites were formed enzymatically or by ROS. Additionally, the ratios of the enantiomers may provide evidence for a specific enzymatic pathway of formation. The use of SID allows for accurate quantification of metabolites and accounts for any sample loss during preparation as well as the differences experienced during ionization. Using the PFB electron capture reagent increases the sensitivity of detection by two orders of magnitude over conventional APCI methods. Overall, this method, SID-LC-EC-atmospheric pressure chemical ionization APCI-MRM/MS, is one of the most sensitive, selective, and accurate methods of quantification for bioactive lipids.

This protocol will demonstrate the extraction and analysis of free and esterified bioactive fatty acids from cells. Fatty acids are accurately quantified using stable isotope dilution, chiral liquid chromatography, electron capture atmospheric chemical ionization multiple reaction monitoring mass spectrometry (SID-LC-ECAPCI-MRM/MS).

The metabolism of fatty acids, such as arachidonic acid (AA) and linoleic acid (LA), results in the formation of oxidized bioactive lipids, including ...

Bacterial Detection &amp; Identification Using Electrochemical Sensors

Electrochemical sensors are widely used for rapid and accurate measurement of blood glucose and can be adapted for detection of a wide variety of analytes. Electrochemical sensors operate by transducing a biological recognition event into a useful electrical signal. Signal transduction occurs by coupling the activity of a redox enzyme to an amperometric electrode. Sensor specificity is either an inherent characteristic of the enzyme, glucose oxidase in the case of a glucose sensor, or a product of linkage between the enzyme and an antibody or probe.
Here, we describe an electrochemical sensor assay method to directly detect and identify bacteria. In every case, the probes described here are DNA oligonucleotides. This method is based on sandwich hybridization of capture and detector probes with target ribosomal RNA (rRNA). The capture probe is anchored to the sensor surface, while the detector probe is linked to horseradish peroxidase (HRP). When a substrate such as 3,3',5,5'-tetramethylbenzidine (TMB) is added to an electrode with capture-target-detector complexes bound to its surface, the substrate is oxidized by HRP and reduced by the working electrode. This redox cycle results in shuttling of electrons by the substrate from the electrode to HRP, producing current flow in the electrode.

We describe an electrochemical sensor assay method for rapid bacterial detection and identification. The assay involves a sensor array functionalized with DNA oligonucleotide capture probes for ribosomal RNA (rRNA) species-specific sequences. Sandwich hybridization of target rRNA with the capture probe and a horseradish peroxidase-linked DNA oligonucleotide detector probe produces a measurable amperometric current.

Electrochemical  sensors are widely used for rapid and accurate measurement of blood glucose and  can be adapted for detection of a wide variety of ...

Imaging of Biological Tissues by Desorption Electrospray Ionization Mass Spectrometry

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological tissues at the hundreds to tens of microns scale. When performed under ambient conditions, sample pre-treatment becomes unnecessary, thus simplifying the protocol while maintaining the high quality of information obtained. Desorption electrospray ionization (DESI) is a spray-based ambient MSI technique that allows for the direct sampling of surfaces in the open air, even in vivo. When used with a software-controlled sample stage, the sample is rastered underneath the DESI ionization probe, and through the time domain, m/z information is correlated with the chemical species' spatial distribution. The fidelity of the DESI-MSI output depends on the source orientation and positioning with respect to the sample surface and mass spectrometer inlet. Herein, we review how to prepare tissue sections for DESI imaging and additional experimental conditions that directly affect image quality. Specifically, we describe the protocol for the imaging of rat brain tissue sections by DESI-MSI.

Desorption electrospray ionization mass spectrometry (DESI-MS) is an ambient method by which samples, including biological tissues, can be imaged with minimal sample preparation. By rastering the sample below the ionization probe, this spray-based technique provides sufficient spatial resolution to discern molecular features of interest within tissue sections.

Mass spectrometry imaging (MSI) provides untargeted molecular information with the highest specificity and spatial resolution for investigating biological ...

Sample Preparation Strategies for Mass Spectrometry Imaging of 3D Cell Culture Models

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with some of the spatial and molecular complexity of tissue. For example, many cell lines form 3D structures given appropriate in vitro conditions. Colon cancer cell lines form 3D cell culture spheroids, in vitro mimics of avascular tumor nodules. While immunohistochemistry and other classical imaging methods are popular for monitoring the distribution of specific analytes, mass spectrometric imaging examines the distribution of classes of molecules in an unbiased fashion. While MALDI mass spectrometric imaging was originally developed to interrogate samples obtained from humans or animal models, this report describes the analysis of in vitro three dimensional cell cultures, including improvements in sample preparation strategies. Herein is described methods for growth, harvesting, sectioning, washing, and analysis of 3D cell cultures via matrix-assisted laser desorption/ionization-mass spectrometry (MALDI-MS) imaging. Using colon carcinoma 3D cell cultures as a model system, this protocol demonstrates the ability to monitor analytes in an unbiased fashion across the 3D cell culture system with MALDI-MSI.

Immortalized cancer cell lines can be grown as 3D cell cultures, a valuable model for biological research. This protocol describes mass spectrometry imaging of 3D cell cultures, including improvements in the sample preparation platform. The goal of this protocol is to instruct users to prepare 3D cell cultures for mass spectrometry imaging analysis.

Three dimensional cell cultures are attractive models for biological research. They combine the flexibility and cost-effectiveness of cell culture with ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization (IR-MALDESI)

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption electrospray ionization (MALDESI) is an example of such methods, where features of matrix-assisted laser desorption/ionization (MALDI) (e.g., pulsed nature of desorption) and electrospray ionization (ESI) (e.g., soft-ionization) are combined. One of the major advantages of MALDESI is its inherent versatility. In MALDESI experiments, an ultraviolet (UV) or infrared (IR) laser can be used to resonantly excite an endogenous or exogenous matrix. The choice of matrix is not analyte dependent, and depends solely on the laser wavelength used for excitation. In IR-MALDESI experiments, a thin layer of ice is deposited on the sample surface as an energy-absorbing matrix. The IR-MALDESI source geometry has been optimized using statistical design of experiments (DOE) for analysis of liquid samples as well as biological tissue specimens. Furthermore, a robust IR-MALDESI imaging source has been developed, where a tunable mid-IR laser is synchronized with a computer controlled XY translational stage and a high resolving power mass spectrometer. A custom graphical user interface (GUI) allows user selection of the repetition rate of the laser, number of shots per voxel, step-size of the sample stage, and the delay between the desorption and scan events for the source. IR-MALDESI has been used in variety of applications such as forensic analysis of fibers and dyes and MSI of biological tissue sections. Distribution of different analytes ranging from endogenous metabolites to exogenous xenobiotics within tissue sections can be measured and quantified using this technique. The protocol presented in this manuscript describes major steps necessary for IR-MALDESI MSI of whole-body tissue sections.

Whole-body Mass Spectrometry Imaging by Infrared Matrix-assisted Laser Desorption Electrospray Ionization IR-MALDESI

A mass spectrometry imaging (MSI) source operated at atmospheric pressure was developed by coupling mid-infrared laser desorption and electrospray post-ionization. Exogenous ice matrix was used as the energy-absorbing matrix to facilitate resonant desorption of tissue-related material. This manuscript provides a step-by-step protocol for performing IR-MALDESI MSI of whole-body neonatal mouse.

Ambient ionization sources for mass spectrometry (MS) have been the subject of much interest in the past decade. Matrix-assisted laser desorption ...

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on two-dimensional surfaces. However, the physiological environment in which in vivo wound healing takes place is three-dimensional rather than two-dimensional. It is becoming increasingly clear that cell behavior differs greatly in two-dimensional vs. three-dimensional environments; therefore, there is a need for more physiologically relevant in vitro models for studying cell migration behaviors in wound closure. The method described herein allows for the study of cell migration in a three-dimensional model that better reflects physiological conditions than previously established two-dimensional scratch assays. The purpose of this model is to evaluate cell outgrowth via the examination of cell migration away from a spheroid body embedded within a fibrin matrix in the presence of pro- or anti-migratory factors. Using this method, cell outgrowth from the spheroid body in a three-dimensional matrix can be observed and is easily quantifiable over time via brightfield microscopy and analysis of spheroid body area. The effect of pro-migratory and/or inhibitory factors on cell migration can also be evaluated in this system. This method provides researchers with a simple method of analyzing cell migration in three-dimensional wound associated matrices in vitro, thus increasing the relevance of in vitro cell studies prior to the use of in vivo animal models.

Characterizing Cell Migration Within Three-dimensional In Vitro Wound Environments

The goal of this protocol is to evaluate the effect of pro- and anti-migratory factors on cell migration within a three-dimensional fibrin matrix.

Currently, most in vitro models of wound healing, such as well-established scratch assays, involve studying cell migration and wound closure on ...

Engineering 'Golden' Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and engineered variants of the green fluorescent protein from Aequorea victoria (avGFP) as well as homologs from other species already cover large parts of the optical spectrum, a spectral gap remains in the near-infrared region, for which avGFP-based fluorophores are not available. Red-shifted fluorescent protein (FP) variants would substantially expand the toolkit for spectral unmixing of multiple molecular species, but the naturally occurring red-shifted FPs derived from corals or sea anemones have lower fluorescence quantum yield and inferior photo-stability compared to the avGFP variants. Further manipulation and possible expansion of the chromophore&#39;s conjugated system towards the far-red spectral region is also limited by the repertoire of 20 canonical amino acids prescribed by the genetic code. To overcome these limitations, synthetic biology can achieve further spectral red-shifting via insertion of non-canonical amino acids into the chromophore triad. We describe the application of SPI to engineer avGFP variants with novel spectral properties. Protein expression is performed in a tryptophan-auxotrophic E. coli strain and by supplementing growth media with suitable indole precursors. Inside the cells, these precursors are converted to the corresponding tryptophan analogs and incorporated into proteins by the ribosomal machinery in response to UGG codons. The replacement of Trp-66 in the enhanced &#34;cyan&#34; variant of avGFP (ECFP) by an electron-donating 4-aminotryptophan results in GdFP featuring a 108 nm Stokes shift and a strongly red-shifted emission maximum (574 nm), while being thermodynamically more stable than its predecessor ECFP. Residue-specific incorporation of the non-canonical amino acid is analyzed by mass spectrometry. The spectroscopic properties of GdFP are characterized by time-resolved fluorescence spectroscopy as one of the valuable applications of genetically encoded FPs in life sciences.

Engineering &#039;Golden&#039; Fluorescence by Selective Pressure Incorporation of Non-canonical Amino Acids and Protein Analysis by Mass Spectrometry and Fluorescence

Synthetic biology enables the engineering of proteins with unprecedented properties using the co-translational insertion of non-canonical amino acids. Here, we presented how a spectrally red-shifted variant of a GFP-type fluorophore with novel fluorescence spectroscopic properties, termed &#34;gold&#34; fluorescent protein (GdFP), is produced in E. coli via selective pressure incorporation (SPI).

Fluorescent proteins are fundamental tools for the life sciences, in particular for fluorescence microscopy of living cells. While wild-type and ...

A Rapid Image-based Bacterial Virulence Assay Using Amoeba

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria can undergo changes in the physiology due to the exposure to host growth environment and the presence of the host cells. We developed an assay to rapidly measure the virulence state of bacteria that minimize the extent to which bacteria grow in the presence of host cells. Bacteria and amoebae are mixed together and immobilized on a single imaging plane using an agar pad. The procedure uses single-cell fluorescence imaging with calcein-acetoxymethyl ester (calcein-AM) as an indicator of host cell health. The fluorescence of host cells is analyzed after 1 h of exposure of host cells to bacteria using epifluorescence microscopy. Image analysis software is used to compute a host killing index. This method has been used to measure virulence within planktonic and surface-attached Pseudomonas aeruginosa sub-populations during the initial stage of biofilm formation and may be adapted to other bacteria and other stages of biofilm growth. This protocol provides a rapid and robust method of measuring virulence and avoids many of the complexities associated with the growth and maintenance of mammalian cell lines. Virulence phenotypes measured here using amoebae have also been validated using mouse macrophages. In particular, this assay was used to establish that surface attachment upregulates virulence in P. aeruginosa.

Here, we present a protocol to measure the virulence of planktonic or surface-attached bacteria using D. discoideum (amoeba) as a host. Virulence is measured over a period of 1 h and host killing is quantified using fluorescence microscopy and image analysis. We demonstrate this protocol using the bacterium P. aeruginosa.

Traditional bacterial virulence assays involve prolonged exposure of bacteria over the course of several hours to host cells. During this time, bacteria ...

Preparing Protein Producing Synthetic Cells using Cell Free Bacterial Extracts, Liposomes and Emulsion Transfer

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell mimicking environment. Furthermore, the development of cell-free expression systems has demonstrated the ability to reconstitute the protein production, transcription and translation processes (DNA&#8594;RNA&#8594;protein) in a controlled manner, harnessing synthetic biology. Here we describe a protocol for preparing a cell-free expression system, including the production of a potent bacterial lysate and encapsulating this lysate inside cholesterol-rich lipid-based giant unilamellar vesicles (GUVs) (i.e., stable liposomes), to form synthetic cells. The protocol describes the methods for preparing the components of the synthetic cells including the production of active bacterial lysates, followed by a detailed step-by-step preparation of the synthetic cells based on a water-in-oil emulsion transfer method. These facilitate the production of millions of synthetic cells in a simple and affordable manner with a high versatility for producing different types of proteins. The obtained synthetic cells can be used to investigate protein/RNA production and activity in an isolated environment, in directed evolution, and also as a controlled drug delivery platform for on-demand production of therapeutic proteins inside the body.

This protocol describes the method, materials, equipment and steps for bottom-up preparation of RNA and protein producing synthetic cells. The inner aqueous compartment of the synthetic cells contained the S30 bacterial lysate encapsulated within a lipid bilayer (i.e., stable liposomes), using a water-in-oil emulsion transfer method.

The bottom-up assembly approach for construction of synthetic cells is an effective tool for isolating and investigating cellular processes in a cell ...