Name: real-time breath analysis by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry
Uploaded: 2018-03-09T12:30:00
Description: Watch this Scientific Journal Video about Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry at JoVE.com

This work has been financially supported by National Natural Science Foundation of China (No. 91543117).

Caution: Please consult all relevant material safety data sheets (MSDS) before use. Please use appropriate personal protective equipment, e.g., lab coat, gloves, goggles, full length pants and closed-toe shoes).
1. Set up the Sec-nanoESI source
<ol>
	<li>Set up a Sec-nanoESI source according to the SESI process, i.e., the breath gas is introduced to intersect an electrospray plume and ionized by the charged droplets (Figure 1). The sources buil...

<ol>
	<li>De Lacy Costello, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+review+of+the+volatiles+from+the+healthy+human+body.">A review of the volatiles from the healthy human body.</a> J. Breath Res. 8 (1), 014001-014030 (2014).</li><li>Phillips, M., Greenberg, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ion-trap+detection+of+volatile+organic+compounds+in+alveolar+breath.">Ion-trap detection of volatile organic compounds in alveolar breath.</a> Clin. Chem. 38 (1), 60-65 (1992).</li><li>Mart&#237;nez-Lozano Sinues, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Circadian+variation+of+the+human+metabolome+captured+by+real-time+breath+analysis.">Circadian variation of the human metabolome captured by real-time breath analysis.</a> PLoS One. 9 (12), 0114422-0114438 (2014).</li><li>Garcia-Gomez, D., et al. <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+coupled+to+high-resolution+mass+spectrometry+reveals+tryptophan+pathway+metabolites+in+exhaled+human+breath.">Secondary electrospray ionization coupled to high-resolution mass spectrometry reveals tryptophan pathway metabolites in exhaled human breath.</a> Chem. Common. 52 (55), 8526-8528 (2016).</li><li>Garcia-Gomez, D., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+quantification+of+amino+acids+in+the+exhalome+by+secondary+electrospray+ionization-mass+spectrometry:+A+proof-of-principle+Study.">Real-time quantification of amino acids in the exhalome by secondary electrospray ionization-mass spectrometry: A proof-of-principle Study.</a> Clin. Chem. 62 (9), 1230-1237 (2016).</li><li>Mart&#237;nez-Lozano Sinues, P., Kohler, M., Brown, S. A., Zenobia, R., Dallmann, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Gauging+circadian+variation+in+ketamine+metabolism+by+real-time+breath+analysis.">Gauging circadian variation in ketamine metabolism by real-time breath analysis.</a> Chem. Common. 53 (14), 2264-2267 (2017).</li><li>Gamez, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time,+in+vivo+monitoring+and+pharmacokinetics+of+valproic+acid+via+a+novel+biomarker+in+exhaled+breath.">Real-time, in vivo monitoring and pharmacokinetics of valproic acid via a novel biomarker in exhaled breath.</a> Chem. Common. 47 (17), 4884-4886 (2011).</li><li>Li, X., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Drug+pharmacokinetics+determined+by+real-time+analysis+of+mouse+breath.">Drug pharmacokinetics determined by real-time analysis of mouse breath.</a> Angew. Chem. Int. Ed. 54 (27), 7815-7818 (2015).</li><li>Amorim, L. L. A., Cardeal, Z. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Breath+air+analysis+and+its+use+as+a+biomarker+in+biological+monitoring+of+occupational+and+environmental+exposure+to+chemical+agents.">Breath air analysis and its use as a biomarker in biological monitoring of occupational and environmental exposure to chemical agents.</a> J. Chromatogr. B. 853 (1-2), 1-9 (2007).</li><li>Bajtarevic, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Noninvasive+detection+of+lung+cancer+by+analysis+of+exhaled+breath.">Noninvasive detection of lung cancer by analysis of exhaled breath.</a> BMC Cancer. 9, 348 (2009).</li><li>Smith, D., Wang, T. S., Pysanenko, A., &#352;pan&#283;l, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+selected+ion+flow+tube+mass+spectrometry+study+of+ammonia+in+mouth-+and+nose-exhaled+breath+and+in+the+oral+cavity.">A selected ion flow tube mass spectrometry study of ammonia in mouth- and nose-exhaled breath and in the oral cavity.</a> Rapid Commun. Mass Spectrom. 22 (6), 783-789 (2008).</li><li>Li, X., Huang, L., Zhu, H., Zhou, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+breath+analysis+by+secondary+nano-electrospray+ionization+ultrahigh+resolution+mass+spectrometry:+Importance+of+high+mass+resolution+and+mass+accuracy.">Direct human breath analysis by secondary nano-electrospray ionization ultrahigh resolution mass spectrometry: Importance of high mass resolution and mass accuracy.</a> Rapid Commun. Mass Spectrom. 31 (3), 301-308 (2017).</li><li>Mart&#237;nez-Lozano, P., Fernandez de la Mora, J. <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 (1), 68-72 (2007).</li><li>Zeng, Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+creatinine+in+exhaled+breath+of+humans+with+chronic+kidney+disease+by+extractive+electrospray+ionization+mass+spectrometry.">Detection of creatinine in exhaled breath of humans with chronic kidney disease by extractive electrospray ionization mass spectrometry.</a> J. Breath Res. 10 (1), 016008-016015 (2016).</li><li>Chen, H. W., Wortmann, A., Zhang, W. H., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+in+vivo+fingerprinting+of+nonvolatile+compounds+in+breath+by+extractive+electrospray+ionization+quadrupole+time-of-flight+mass+spectrometry.">Rapid in vivo fingerprinting of nonvolatile compounds in breath by extractive electrospray ionization quadrupole time-of-flight mass spectrometry.</a> Angew. Chem. Int. Ed. 46 (4), 580-583 (2007).</li><li>Benoi, F. M., Davldson, W. R., Lovett, A. M., Nacson, S., Ngo, A. <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+by+atmospheric+pressure+ionization+mass+spectrometry.">Breath analysis by atmospheric pressure ionization mass spectrometry.</a> Anal. Chem. 55 (4), 805-807 (1983).</li><li>Bregy, L., Mart&#237;nez-Lozano Sinues, P., Nudnova, M. M., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Real-time+breath+analysis+with+active+capillary+plasma+ionization-ambient+mass+spectrometry.">Real-time breath analysis with active capillary plasma ionization-ambient mass spectrometry.</a> J. Breath Res. 8 (2), 027102-027110 (2014).</li><li>Gaugg, M. T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+metabolite+coverage+of+real-time+breath+analysis+by+coupling+a+universal+secondary+electrospray+ionization+source+and+high+resolution+mass+spectrometry-a+pilot+study+on+tobacco+smokers.">Expanding metabolite coverage of real-time breath analysis by coupling a universal secondary electrospray ionization source and high resolution mass spectrometry-a pilot study on tobacco smokers.</a> J. Breath Res. 10 (1), 016010-016020 (2016).</li><li>Mart&#237;nez-Lozano, P., Zingaro, L., Finiguerra, A., Cristoni, S. <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-mass+spectrometry:+breath+study+on+a+control+group.">Secondary electrospray ionization-mass spectrometry: breath study on a control group.</a> J. Breath Res. 5 (1), 016002-016012 (2011).</li><li>Mart&#237;nez-Lozano Sinues, P., Zenobi, R., Kohler, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Analysis+of+the+exhalome+a+diagnostic+tool+of+the+future.">Analysis of the exhalome a diagnostic tool of the future.</a> Chest. 144 (3), 746-749 (2013).</li><li>Mart&#237;nez-Lozano Sinues, P., Fernandez de la Mora, 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 (21), 8210-8215 (2008).</li><li>Mart&#237;nez-Lozano Sinues, P., et al. <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+in+real+time+by+mass+spectrometry+in+chronic+obstructive+pulmonary+disease.">Breath analysis in real time by mass spectrometry in chronic obstructive pulmonary disease.</a> Respiration. 87 (4), 301-310 (2014).</li><li>Mart&#237;nez-Lozano Sinues, P., Kohler, M., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Monitoring+diurnal+changes+in+exhaled+human+breath.">Monitoring diurnal changes in exhaled human breath.</a> Anal. Chem. 85 (1), 369-373 (2013).</li><li>Chen, H. W., Zenobi, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Neutral+desorption+sampling+of+biological+surfaces+for+rapid+chemical+characterization+by+extractive+electropray+ionization+mass+spectrometry.">Neutral desorption sampling of biological surfaces for rapid chemical characterization by extractive electropray ionization mass spectrometry.</a> Nat. Protoc. 3 (9), 1467-1475 (2008).</li><li>Li, X., Hu, B., Ding, J., Chen, H. W. <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+samples+at+molecular+levels+by+neutral+desorption+extractive+electrospray+ionization+mass+spectrometry.">Rapid characterization of complex viscous samples at molecular levels by neutral desorption extractive electrospray ionization mass spectrometry.</a> Nat. Protoc. 7 (6), 1010-1025 (2011).</li><li>Gordon, S. M., Szidon, J. P., Krotoszynski, B. K., Gibbons, R. D., O'Neill, H. J. <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+exhaled+air+from+patients+with+lung+cancer.">Volatile organic compounds in exhaled air from patients with lung cancer.</a> Clin. Chem. 31 (8), 1278-1282 (1985).</li><li>Ding, J. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+of+extractive+electrospray+ionization+ion+trap+mass+spectrometry+in+vivo+breath+analysis.">Development of extractive electrospray ionization ion trap mass spectrometry in vivo breath analysis.</a> Analyst. 134 (10), 2040-2050 (2009).</li><li>Basum, G., Dahnke, H., Halmer, D., Hering, P., M&#252;rtz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+recording+of+ethane+trances+in+human+breath+via+infrared+laser+spectroscopy.">Online recording of ethane trances in human breath via infrared laser spectroscopy.</a> J. Appl. Physiol. 95 (6), 2583-2590 (2003).</li><li>T&#248;ien, &. #. 2. 1. 6. ;. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Automated+open+flow+respirometry+in+continuous+and+long-term+measurements:+design+and+principles.">Automated open flow respirometry in continuous and long-term measurements: design and principles.</a> J. Appl. Physiol. 114 (8), 1094-1107 (2013).</li><li>Huang, L., Li, X., Xu, M., Huang, Z. X., Zhou, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Identification+of+relatively+high+molecular+weight+compounds+in+human+breath+using+secondary+nano+electrospray+ionization+ultrahigh+resolution+mass+spectrometry.">Identification of relatively high molecular weight compounds in human breath using secondary nano electrospray ionization ultrahigh resolution mass spectrometry.</a> Chem. J. Chinese U. 38 (5), 752-757 (2017).</li></ol>

Constructing the Sec-nanoESI source based on a commercial nanoESI source, the ionization efficiency is higher than that of using an ESI source30. In addition, the ionization efficiency is further improved in a closed chamber, as it isolates the process from the ambient background air, and at the same time facilitates the mixing between the gas sample and the spray plume. By using a Sec-nanoESI, less parameters need to be optimized compared to an ESI source, making it easier for installation, appli...

With fast development of modern analytical techniques, hundreds of volatile organic compounds (VOCs) have been identified in human exhaled breath1. These VOCs mostly result from alveolar air (~350 mL for a healthy adult) and anatomical dead space air (~150 mL)2, which are affected by body metabolism3,4,5,6,7,8 and environmental pollution9, respectively. As a result, if identified, these VOCs are promising to be used as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner.
Though gas chromatography mass spectrometry (GC-MS) is the most widely used technique for qualitative and quantitative analysis of exhaled VOCs2, direct MS techniques, which have been developed for real-time breath analysis, have the advantages of high time resolution and simple sample pre-preparation. Direct MS techniques, such as proton transfer reaction MS (PTR-MS)10, selected ion flow tube MS (SIFT-MS)11, secondary electrospray ionization MS (SESI-MS)12,13 (also named as extractive electrospray ionization MS, EESI-MS14,15), trace atmospheric gas analyzer (TAGA)16 and plasma ionization MS (PI-MS)17 have been investigated in recent years.
Among all the direct MS techniques, SESI is well-known as a universal soft ionization technique19,20,21; and the source is easy to be customized and coupled to different types of mass spectrometers, e.g., time of flight mass spectrometer8,15, ion trap mass spectrometer14 and orbitrap mass spectrometer12,18. Up to now, SESI-MS has been successfully used in diagnosing respiratory diseases22, gauging circadian rhythm3,6,23, pharmacokinetics7,8, and revealing metabolic pathways4, etc. Most recently, a commercial SESI source has become available.
In this study, a facile and compact secondary nanoelectrospray ionization source (Sec-nanoESI) was set up and coupled to a high-resolution mass spectrometer. Real-time measurements of exhaled VOCs in breath were presented.

<table>
	<tbody>
		<tr>
			<td>Ultrapure water</td>
			<td>Merck Millipore, USA</td>
			<td>MPGP04001</td>
			<td>Resistance &#62;18.2 M&#937;&#183;cm</td>
		</tr>
		<tr>
			<td>Formic acid</td>
			<td>Sigma-Aldrich, USA</td>
			<td>F0507</td>
			<td>Corrosive to the respiratory tract.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>Guangzhou Shiyuan Gas Co. Ltd., China</td>
			<td>N.A.a</td>
			<td>Purity &#62;99.99%</td>
		</tr>
		<tr>
			<td>Q Exactive hybrid quadrupole-orbitrap mass spectrometer</td>
			<td>Thermo Scientific, USA</td>
			<td>02634L(S/N)</td>
			<td>Beware of high voltage and high temperature</td>
		</tr>
		<tr>
			<td>NanoESI source</td>
			<td>Thermo Scientific, USA</td>
			<td>ES002373(S/N); ES071(P/N)</td>
			<td>Beware of high voltage and high temperature</td>
		</tr>
		<tr>
			<td>Nano LC pump</td>
			<td>Thermo Scientific, USA</td>
			<td>5041.0010A(P/N)</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Xcalibur software (Version 3.0)</td>
			<td>Thermo Scientific, USA</td>
			<td>BRE0008596</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Dino-Lite Digital Microscope</td>
			<td>Tech Video System (SuZhou) Co.Ltd., China</td>
			<td>CQ401833R(S/N)</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Nafion tubing</td>
			<td>Perma Pure LLC, USA</td>
			<td>ME60</td>
			<td>/</td>
		</tr>
		<tr>
			<td>PTFE tubing (I.D. 4 mm)</td>
			<td>Dongguan Hongfu Insulating Material Co. Ltd., China</td>
			<td>N.A.</td>
			<td>Beware of the possible loss of polar compounds</td>
		</tr>
		<tr>
			<td>Mass flow controller</td>
			<td>Line-Tech, Korea</td>
			<td>M15122007 (S/N)</td>
			<td>/</td>
		</tr>
		<tr>
			<td>Flow meter</td>
			<td>Yuyao Industrial Automation Meter Factory, China</td>
			<td>40784</td>
			<td>/</td>
		</tr>
		<tr>
			<td>aN.A.: not available.</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

real-time breath analysis by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental exposure in a non-invasive manner. In this work, we present a protocol to characterize the exhaled VOCs in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry (Sec-nanoESI-HRMS). The homemade Sec-nanoESI source was readily set up based on a commercial nanoESI source. Hundreds of peaks were observed in the background-subtracted mass spectra of exhaled breath, and the mass accuracy values are -4.0-13.5 ppm and -20.3-1.3 ppm in the positive and negative ion detection modes, respectively. The peaks were assigned with accurate elemental composition according to the accurate mass and isotopic pattern. Less than 30 s is used for one exhalation measurement, and it takes approximately 7 min for six replicated measurements.

Figure 3 shows the breath fingerprints in the mass range of m/z 50-750 recorded under both positive and negative ion detection modes. 291 peaks (peak intensity &#62; 5.0x104) and 173 peaks (peak intensity &#62; 3.0x104) have been observed in background-subtracted breath fingerprints in the positive and negative ion detection modes, respectively. To identify peaks in the mass spectra, please refer to prior publications for detail...

Watch this Scientific Journal Video about Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry at JoVE.com

Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry

A protocol for characterizing chemical composition of exhaled breath in real time by using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry is demonstrated.

Exhaled volatile organic compounds (VOCs) have aroused considerable interest, since they can serve as biomarkers for disease diagnosis and environmental ...

The overall goal of this experiment is to characterize chemical composition of exhaled breath using secondary nanoelectrospray ionization coupled to high resolution mass spectrometry. This method can help to answer key questions in the medical field such as early diagnosis of diseases, therapeutic check monitoring, and environmental exposure. The main advantage of the technique is the measurement can be performed in real time.The technique is also highest output, sensitive, specific, and user friendly. The implications of this technique have been extend to the diagnosis of lung cancer. Because chance of bio marker candidates have been find in exhaled breath of lung caner patients at different stages.Generally, individuals new to this method will struggle because the ionization source is commonly co-made. Visual demonstration is critical, as there is no standard protocol for this method yet. To begin, set up a secondary nano ESI source according to the secondary electrospary ionization process in which breath gas is introduced to intersect an electrospray plume and ionized by the charged droplets.The sources for the individual labs depend on the mass spectrometer used. Here, the secondary nano ESI source is based on the commercial nano ESI source. And attached it to a bench drop quarter pull orbit trap mass spectrometer.The main body of the source is a cubic stainless steel chamber with an inlet to introduce the nano ESI capillary into the chamber. Two stainless steel tubes are installed on each side of the chamber for gas delivery. Two quartz windows are equipped on the top and bottom of the chamber to check the position of the tip of the nano ESI capillary and nano ESI spray either by eyes or a digital microscope.Finally, the chamber is welded to the sweep cone of the mass spectrometer. Note that the design may change depending on the particular geometry of the atmospheric pressure interface of the mass spectrometer used in individual labs. Calibrate the mass spectrometer in both positive and negative ion detection modes according to manufacturers instructions.By applying calibration, mass spectrometer parameters, such as lens potentials and detection conditions are optimized to give good sensitivity and peak shape at a specified resolution value. Perform complete calibrations by using the commercial ESI source. The mass resolution of 70, 000 is used here.Mass calibration can be performed with any compatible sources, including customized ones. Set the temperature of the ion transfer tube of the mass spectrometer to greater than 100 degrees Celsius. Though the highest temperature can be set at 350 degrees Celsius, it may result in the decomposition of some compounds.For the ESI solvent and flow rate, select the appropriate ESI solvent on the basis of properties of solvent and targeted compounds. In this experiment use water containing 0.1%Formic Acid, for high ionization efficiency. Set the flow rate of the ESI solvent in the range of 0 1.5 microliters per minute, to 200 nanoliters per minute.Optimize the secondary nano ESI source parameters, mainly nano ESI voltage and nano ESI capillary tip position. The voltage commonly ranges from 2.0 4.5 kilovolts. Use 2.5 kilovolts here.Apply pure gas to the source to reduce the influence of volatile organic compounds or VOCs from indoor air. High purity nitrogen is used here and delivered at 0.8 liters per minute. With the presence of pure gas, the normalized intensity level observed in a mass spectrum should be greater than 1 x 10 to the 5th, and the variation of the TIC should be less than 10%in both positive and negative ion detection modes.When performing measurement, inhale the indoor air and perform a normal exhalation to breathe out all the air in the lungs at a constant flow rate. Monitor the exhalation flow rate either by a manometer or a flow meter visible to the subject. Connect the inlet of the flow meter to nafion tubing to remove the water vapor in the exhaled breath.Then, connect the outlet of the flow meter to PTFE tubing to deliver the breath gas. In this experiment, the subject exhaled at 0.4 liters per minute, as controlled by a flow meter. It takes less than 30 seconds for one exhalation measurement.Perform four to six replicated measurements. To minimize confounding effects, have participants abstain from eating, drinking, and brushing their teeth at least 30 minutes prior to the measurements. During the measurement, keep checking if the iron intensity exceeds the linear detection limit of the instrument or not.The saturation of signal can lead to an artifact peak that does not result from a compound in the sample. By inhaling through the nose, part of ambient VOCs and particles would be removed. However, it is noteworthy that compounds in the nasal passages may also be detected.Use software to record chromatagrams and mass spectra. Because this is direct MS analysis, and no chromatagraphic separation is performed, the TIC actually indicates the time trace of all the signals detected in the mass spectra. And the EIC shows a time trace of a specified compound.Shown here are representative results of exhaled breath measurements in real time. The time trace of endol, a typical endogenous compound, shows six replicated measurements in less than seven minutes. For all the measurements, reproducible background subtracted breath fingerprints were obtained in positive ion detection mode.291 compounds have been observed. The compounds are most likely aldehydes, ketones, and alkynes. In the case of breath fingerprints in negative ion detection mode, 173 compounds are presented, and mainly result from fatty acids.After watching this video, you should have a good understanding of how to set up a homemade CESI source and perform real time process analysis. Once mastered, the measurement can be performed in minutes. If it is performed properly.While attending this procedure, it is important to remember to avoid interference of the compounds from the sampling tubing and endo air. Know this measure can provide insights into human exhaled breath, and can also be applied to other systems such as model alkylize, ion models, and in retro-cell systems. Following this procedure, other measures like GCMS, HPRCMS, can be performed to answer additional questions, like for photo confirmation, quantity of analysis.After it's development, the technique paved the ways for researchers in the field of medicine to explore with this diagnosis, treatment effects in the patients.

real-time-breath-analysis-using-secondary-nanoelectrospray-ionization

Research

JoVE Journal

Chemistry

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

A Study of the Complexation of Mercury(II) with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, by electrospray ionization orbitrap mass spectrometry. This strategy is based on previous successful characterization of mercury-dicysteinyl complexes involving tripeptides by utilizing mass spectrometry among other techniques. Mercury(II) chloride and a dicysteinyl tetrapeptide were incubated in a degassed buffered medium at varying stoichiometric ratios. The complexes formed were subsequently analyzed on an electrospray mass spectrometer consisting of a hybrid linear ion- and orbi- trap mass analyzer. The electrospray ionization mass spectrometry (ESI-MS) spectra were acquired in the positive mode and the observed peaks were then analyzed for distinct mercury isotopic distribution patterns and associated monoisotopic peak. This work demonstrates that an accurate stoichiometry of mercury and peptide in the complexes formed under specified electrospray ionization conditions can be determined by using high resolution ESI MS based on distinct mercury isotopic distribution patterns.

A Study of the Complexation of MercuryII with Dicysteinyl Tetrapeptides by Electrospray Ionization Mass Spectrometry

The characterization of complexes formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptides by electrospray ionization orbitrap mass spectrometry is presented.

In this study we evaluated a method for the characterization of complexes, formed in different relative ratios of mercury(II) to dicysteinyl tetrapeptide, ...

Rapid High-throughput Species Identification of Botanical Material Using Direct Analysis in Real Time High Resolution Mass Spectrometry

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, and that these chemical fingerprints can be used for plant species identification. The mass spectral data can be acquired rapidly and in a high throughput manner without the need for sample extraction, derivatization or pH adjustment steps. The use of this technique bypasses challenges presented by more conventional techniques including lengthy chromatography analysis times and resource intensive methods. The high throughput capabilities of the direct analysis in real time-high resolution mass spectrometry protocol, coupled with multivariate statistical analysis processing of the data, provide not only class characterization of plants, but also yield species and varietal information. Here, the technique is demonstrated with two psychoactive plant products, Mitragyna speciosa (Kratom) and Datura (Jimsonweed), which were subjected to direct analysis in real time-high resolution mass spectrometry followed by statistical analysis processing of the mass spectral data. The application of these tools in tandem enabled the plant materials to be rapidly identified at the level of variety and species.

A method for species identification of botanical material by direct analysis in real time-high resolution mass spectrometry and multivariate statistical analysis is presented.

We demonstrate that direct analysis in real time-high resolution mass spectrometry can be used to produce mass spectral profiles of botanical material, ...

Fizzy Extraction of Volatile Organic Compounds Combined with Atmospheric Pressure Chemical Ionization Quadrupole Mass Spectrometry

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to the gas phase, and efficiently transferred to a detection system. Fizzy extraction takes advantage of the effervescence phenomenon. First, a carrier gas (here, carbon dioxide) is dissolved in the sample by applying overpressure and stirring the sample. Second, the sample chamber is decompressed abruptly. Decompression leads to the formation of numerous carrier gas bubbles in the sample liquid. These bubbles assist the release of the dissolved analyte species from the liquid to the gas phase. The released analytes are immediately transferred to the atmospheric pressure chemical ionization interface of a triple quadrupole mass spectrometer. The ionizable analyte species give rise to mass spectrometric signals in the time domain. Because the release of the analyte species occurs over short periods of time (a few seconds), the temporal signals have high amplitudes and high signal-to-noise ratios. The amplitudes and areas of the temporal peaks can then be correlated with concentrations of the analytes in the liquid samples subjected to fizzy extraction, which enables quantitative analysis. The advantages of fizzy extraction include: simplicity, speed, and limited use of chemicals (solvents).

Fizzy extraction is a new laboratory technique for analysis of volatile and semivolatile compounds. A carrier gas is dissolved in the liquid sample by applying overpressure and stirring the sample. The sample chamber is then decompressed. The analyte species are liberated to the gas phase due to effervescence.

Chemical analysis of volatile and semivolatile compounds dissolved in liquid samples can be challenging. The dissolved components need to be brought to ...

Synthesis and Mass Spectrometry Analysis of Oligo-peptoids

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have been thought of as a type of molecular information storage. Mass spectrometry analysis has been considered the method of choice for sequencing peptoids. Peptoids can be synthesized via solid phase chemistry using a repeating two-step reaction cycle. Here we present a method to manually synthesize oligo-peptoids and to analyze the sequence of the peptoids using tandem mass spectrometry (MS/MS) techniques. The sample peptoid is a nonamer consisting of alternating N-(2-methyloxyethyl)glycine (Nme) and N-(2-phenylethyl)glycine (Npe), as well as an N-(2-aminoethyl)glycine (Nae) at the N-terminus. The sequence formula of the peptoid is Ac-Nae-(Npe-Nme)4-NH2, where Ac is the acetyl group. The synthesis takes place in a commercially available solid-phase reaction vessel. The rink amide resin is used as the solid support to yield the peptoid with an amide group at the C-terminus. The resulting peptoid product is subjected to sequence analysis using a triple-quadrupole mass spectrometer coupled to an electrospray ionization source. The MS/MS measurement produces a spectrum of fragment ions resulting from the dissociation of charged peptoid. The fragment ions are sorted out based on the values of their mass-to-charge ratio (m/z). The m/z values of the fragment ions are compared against the nominal masses of theoretically predicted fragment ions, according to the scheme of peptoid fragmentation. The analysis generates a fragmentation pattern of the charged peptoid. The fragmentation pattern is correlated to the monomer sequence of the neutral peptoid. In this regard, MS analysis reads out the sequence information of the peptoids.

A protocol is described for the manual synthesis of oligo-peptoids followed by sequence analysis by mass spectrometry.

Peptoids are sequence-controlled peptide-mimicking oligomers consisting of N-alkylated glycine units. Among many potential applications, peptoids have ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Imaging Corrosion at the Metal-Paint Interface Using Time-of-Flight Secondary Ion Mass Spectrometry

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass spectrometry (ToF-SIMS), illustrating that SIMS is a suitable technique to study the chemical distribution at a metal-paint interface. The painted Al alloy coupons are immersed in a salt solution or exposed to air only. SIMS provides chemical mapping and 2D molecular imaging of the interface, allowing direct visualization of the morphology of the corrosion products formed at the metal-paint interface and mapping of the chemical after corrosion occurs. The experimental procedure of this method is presented to provide technical details to facilitate similar research and highlight pitfalls that may be encountered during such experiments.

Time-of-flight secondary ion mass spectrometry is applied to demonstrate the chemical mapping and corrosion morphology at the metal-paint interface of an aluminum alloy after being exposed to a salt solution compared with a specimen exposed to air.

Corrosion developed at the paint and aluminum (Al) metal-paint interface of an aluminum alloy is analyzed using time-of-flight secondary ion mass ...

Analysis of Complex Molecules and Their Reactions on Surfaces by Means of Cluster-Induced Desorption/Ionization Mass Spectrometry

Desorption/Ionization Induced by Neutral SO2 Clusters (DINeC) is employed as a very soft and efficient desorption/ionization technique for mass spectrometry (MS) of complex molecules and their reactions on surfaces. DINeC is based on a beam of SO2 clusters impacting on the sample surface at low cluster energy. During cluster-surface impact, some of the surface molecules are desorbed and ionized via dissolvation in the impacting cluster; as a result of this dissolvation-mediated desorption mechanism, low cluster energy is sufficient and the desorption process is extremely soft. Both surface adsorbates and molecules of which the surface is composed of can be analyzed. Clear and fragmentation-free spectra from complex molecules such as peptides and proteins are obtained. DINeC does not require any special sample preparation, in particular no matrix has to be applied. The method yields quantitative information on the composition of the samples; molecules at a surface coverage as low as 0.1 % of a monolayer can be detected. Surface reactions such as H/D exchange or thermal decomposition can be observed in real-time and the kinetics of the reactions can be deduced. Using a pulsed nozzle for cluster beam generation, DINeC can be efficiently combined with ion trap mass spectrometry. The matrix-free and soft nature of the DINeC process in combination with the MSn capabilities of the ion trap allows for very detailed and unambiguous analysis of the chemical composition of complex organic samples and organic adsorbates on surfaces.

Neutral SO2 clusters of low kinetic energy (&#60; 0.8 eV/constituent) are used to desorb complex surface molecules such as peptides or lipids for further analysis by means of mass spectrometry using an ion trap mass spectrometer. No special sample preparation is required, and real-time observation of reactions is possible.