利用二次 Nanoelectrospray 电离耦合高分辨质谱的实时呼吸分析
Summary
本文证明了用二次 nanoelectrospray 电离耦合高分辨质谱法对呼气的化学成分进行实时表征的一种协议。
Abstract
呼出挥发性有机化合物 (voc) 引起了相当大的兴趣, 因为它们可以作为疾病诊断和环境暴露的生物标志物, 以无创的方式。在这项工作中, 我们提出了一个协议, 用二次 nanoelectrospray 电离耦合到高分辨率质谱 (Sec-nanoESI-HRMS) 实时表征呼出的挥发性有机化合物。自制的 Sec nanoESI 源很容易建立在商业 nanoESI 的基础上。在呼气的背景下减去的质谱中观察到数以百计的峰值, 其质量准确度分别为正负离子检测模式的 4.0-13.5 ppm 和-20.3-1.3 ppm。根据精确的质量和同位素模式, 对峰进行了精确的元素组成。少于三十年代用于一次呼气测量, 并且它需要大约7分钟为六个被复制的测量。
Introduction
随着现代分析技术的飞速发展, 数以百计的挥发性有机化合物 (voc) 已经在人呼出的呼吸中发现了1。这些挥发气主要是由肺泡空气 (350 毫升为一个健康的成人) 和解剖死亡空间空气 (~ 150 毫升)2, 受身体新陈代谢的影响3,4,5,6,7 ,8和环境污染9分别。因此, 如果确定, 这些挥发性有机化合物有望被用作疾病诊断和环境暴露的生物标志物。
气相色谱质谱法 (gc-ms) 是对呼出挥发性有机化合物进行定性定量分析的最广泛应用的技术2, 直接 MS 技术已经发展为实时呼吸分析, 具有高的时间分辨率和简单的样品预准备。直接 MS 技术, 如质子转移反应 ms (PTR)10, 选定的离子流管 ms (筛-ms)11, 二次电喷雾电离 ms (局)12,13 (也命名为萃取电喷雾电离 ms, EESI14,15), 微量大气气体分析仪 (TAGA)16和等离子电离 ms (PI-毫秒)17已被调查近年来。
在所有的直接 MS 技术中, 局是众所周知的通用软电离技术19,20,21;源很容易被定制, 并结合到不同类型的质谱仪,例如, 飞行质量光谱仪的时间8,15, 离子阱质谱仪14和 orbitrap 质谱仪12 ,18。到目前为止, 局已成功地用于诊断呼吸系统疾病22, 测量昼夜节律3,6,23, 药代动力学7,8, 和揭示代谢通路4,等。最近, 一个商业局源已成为可用。
在本研究中, 建立了一种轻便紧凑的二次 nanoelectrospray 电离源 (Sec nanoESI), 并将其耦合到高分辨率质谱仪中。对呼出的挥发性有机化合物进行了实时测量。
Protocol
警告: 使用前请查阅所有相关的材料安全数据表 (MSDS)。请使用适当的个人防护设备,例如, 实验室外套, 手套, 护目镜, 全长长裤和闭合脚趾鞋)。 1. 设置 Sec-nanoESI 源 根据局过程设置 Sec nanoESI 源,即, 引入呼吸气体, 使其与电喷雾羽流相交, 并被带电的水滴电离 (图 1)。在单个实验室中生成的源取决于使用了24、<s…
Representative Results
图 3显示了在正负离子检测模式下记录的m/z 50-750 的质量范围内的呼吸指纹。分别在正负离子检测模式下, 观察了291峰 (峰值强度 > 5.0×104) 和173峰值 (峰值强度 > 3.0×104)。要识别质量谱中的峰值, 请参阅以前发布的详细信息12,18,24,29。?…
Discussion
建立基于商业 nanoESI 源的 Sec-nanoESI 源, 其电离效率高于使用 ESI 源30。此外, 封闭腔内的电离效率进一步提高, 因为它将过程与环境背景空气隔离开来, 同时促进气体样品与喷雾羽流的混合。通过使用 Sec nanoESI, 相对于 ESI 源, 需要优化较少的参数, 从而便于安装、应用和维护。
如果 nanoESI-MS 进行呼吸分析时没有观察到信号或灵敏度显著下降, 应检查喷雾毛细管尖…
Divulgations
The authors have nothing to disclose.
Acknowledgements
这项工作得到了中国国家自然科学基金 (91543117 号) 的资助。
Materials
| Ultrapure water | Merck Millipore, USA | MPGP04001 | Resistance >18.2 MΩ·cm |
| Formic acid | Sigma-Aldrich, USA | F0507 | Corrosive to the respiratory tract. |
| Nitrogen gas | Guangzhou Shiyuan Gas Co. Ltd., China | N.A.a | Purity >99.99% |
| Q Exactive hybrid quadrupole-orbitrap mass spectrometer | Thermo Scientific, USA | 02634L(S/N) | Beware of high voltage and high temperature |
| NanoESI source | Thermo Scientific, USA | ES002373(S/N); ES071(P/N) | Beware of high voltage and high temperature |
| Nano LC pump | Thermo Scientific, USA | 5041.0010A(P/N) | / |
| Xcalibur software (Version 3.0) | Thermo Scientific, USA | BRE0008596 | / |
| Dino-Lite Digital Microscope | Tech Video System (SuZhou) Co.Ltd., China | CQ401833R(S/N) | / |
| Nafion tubing | Perma Pure LLC, USA | ME60 | / |
| PTFE tubing (I.D. 4 mm) | Dongguan Hongfu Insulating Material Co. Ltd., China | N.A. | Beware of the possible loss of polar compounds |
| Mass flow controller | Line-Tech, Korea | M15122007 (S/N) | / |
| Flow meter | Yuyao Industrial Automation Meter Factory, China | 40784 | / |
| aN.A.: not available. |
References
- De Lacy Costello, B., et al. A review of the volatiles from the healthy human body. J. Breath Res. 8 (1), 014001-014030 (2014).
- Phillips, M., Greenberg, J. Ion-trap detection of volatile organic compounds in alveolar breath. Clin. Chem. 38 (1), 60-65 (1992).
- Martínez-Lozano Sinues, P., et al. Circadian variation of the human metabolome captured by real-time breath analysis. PLoS One. 9 (12), 0114422-0114438 (2014).
- Garcia-Gomez, D., et al. Secondary electrospray ionization coupled to high-resolution mass spectrometry reveals tryptophan pathway metabolites in exhaled human breath. Chem. Common. 52 (55), 8526-8528 (2016).
- Garcia-Gomez, D., et al. Real-time quantification of amino acids in the exhalome by secondary electrospray ionization-mass spectrometry: A proof-of-principle Study. Clin. Chem. 62 (9), 1230-1237 (2016).
- Martínez-Lozano Sinues, P., Kohler, M., Brown, S. A., Zenobia, R., Dallmann, R. Gauging circadian variation in ketamine metabolism by real-time breath analysis. Chem. Common. 53 (14), 2264-2267 (2017).
- Gamez, G., et al. Real-time, in vivo monitoring and pharmacokinetics of valproic acid via a novel biomarker in exhaled breath. Chem. Common. 47 (17), 4884-4886 (2011).
- Li, X., et al. Drug pharmacokinetics determined by real-time analysis of mouse breath. Angew. Chem. Int. Ed. 54 (27), 7815-7818 (2015).
- Amorim, L. L. A., Cardeal, Z. L. Breath air analysis and its use as a biomarker in biological monitoring of occupational and environmental exposure to chemical agents. J. Chromatogr. B. 853 (1-2), 1-9 (2007).
- Bajtarevic, A., et al. Noninvasive detection of lung cancer by analysis of exhaled breath. BMC Cancer. 9, 348 (2009).
- Smith, D., Wang, T. S., Pysanenko, A., Španěl, P. A selected ion flow tube mass spectrometry study of ammonia in mouth- and nose-exhaled breath and in the oral cavity. Rapid Commun. Mass Spectrom. 22 (6), 783-789 (2008).
- Li, X., Huang, L., Zhu, H., Zhou, Z. Direct human breath analysis by secondary nano-electrospray ionization ultrahigh resolution mass spectrometry: Importance of high mass resolution and mass accuracy. Rapid Commun. Mass Spectrom. 31 (3), 301-308 (2017).
- Martínez-Lozano, P., Fernandez de la Mora, J. Electrospray ionization of volatiles in breath. Int. J. Mass Spectrom. 265 (1), 68-72 (2007).
- Zeng, Q., et al. Detection of creatinine in exhaled breath of humans with chronic kidney disease by extractive electrospray ionization mass spectrometry. J. Breath Res. 10 (1), 016008-016015 (2016).
- Chen, H. W., Wortmann, A., Zhang, W. H., Zenobi, R. Rapid in vivo fingerprinting of nonvolatile compounds in breath by extractive electrospray ionization quadrupole time-of-flight mass spectrometry. Angew. Chem. Int. Ed. 46 (4), 580-583 (2007).
- Benoi, F. M., Davldson, W. R., Lovett, A. M., Nacson, S., Ngo, A. Breath analysis by atmospheric pressure ionization mass spectrometry. Anal. Chem. 55 (4), 805-807 (1983).
- Bregy, L., Martínez-Lozano Sinues, P., Nudnova, M. M., Zenobi, R. Real-time breath analysis with active capillary plasma ionization-ambient mass spectrometry. J. Breath Res. 8 (2), 027102-027110 (2014).
- Gaugg, M. T., et al. 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. J. Breath Res. 10 (1), 016010-016020 (2016).
- Martínez-Lozano, P., Zingaro, L., Finiguerra, A., Cristoni, S. Secondary electrospray ionization-mass spectrometry: breath study on a control group. J. Breath Res. 5 (1), 016002-016012 (2011).
- Martínez-Lozano Sinues, P., Zenobi, R., Kohler, M. Analysis of the exhalome a diagnostic tool of the future. Chest. 144 (3), 746-749 (2013).
- Martínez-Lozano Sinues, P., Fernandez de la Mora, J. Direct analysis of fatty acid vapors in breath by electrospray ionization and atmospheric pressure Ionization-Mass Spectrometry. Anal. Chem. 80 (21), 8210-8215 (2008).
- Martínez-Lozano Sinues, P., et al. Breath analysis in real time by mass spectrometry in chronic obstructive pulmonary disease. Respiration. 87 (4), 301-310 (2014).
- Martínez-Lozano Sinues, P., Kohler, M., Zenobi, R. Monitoring diurnal changes in exhaled human breath. Anal. Chem. 85 (1), 369-373 (2013).
- Chen, H. W., Zenobi, R. Neutral desorption sampling of biological surfaces for rapid chemical characterization by extractive electropray ionization mass spectrometry. Nat. Protoc. 3 (9), 1467-1475 (2008).
- Li, X., Hu, B., Ding, J., Chen, H. W. Rapid characterization of complex viscous samples at molecular levels by neutral desorption extractive electrospray ionization mass spectrometry. Nat. Protoc. 7 (6), 1010-1025 (2011).
- Gordon, S. M., Szidon, J. P., Krotoszynski, B. K., Gibbons, R. D., O’Neill, H. J. Volatile organic compounds in exhaled air from patients with lung cancer. Clin. Chem. 31 (8), 1278-1282 (1985).
- Ding, J. H., et al. Development of extractive electrospray ionization ion trap mass spectrometry in vivo breath analysis. Analyst. 134 (10), 2040-2050 (2009).
- Basum, G., Dahnke, H., Halmer, D., Hering, P., Mürtz, M. Online recording of ethane trances in human breath via infrared laser spectroscopy. J. Appl. Physiol. 95 (6), 2583-2590 (2003).
- Tøien, &. #. 2. 1. 6. ;. Automated open flow respirometry in continuous and long-term measurements: design and principles. J. Appl. Physiol. 114 (8), 1094-1107 (2013).
- Huang, L., Li, X., Xu, M., Huang, Z. X., Zhou, Z. Identification of relatively high molecular weight compounds in human breath using secondary nano electrospray ionization ultrahigh resolution mass spectrometry. Chem. J. Chinese U. 38 (5), 752-757 (2017).
Citer Cet Article
Li, X., Huang, D. D., Du, R., Zhang, Z. J., Chan, C. K., Huang, Z. X., Zhou, Z. Real-time Breath Analysis by Using Secondary Nanoelectrospray Ionization Coupled to High Resolution Mass Spectrometry. J. Vis. Exp. (133), e56465, doi:10.3791/56465 (2018).