School of Engineering, University of Vermont
Bean, H. D., Zhu, J., Hill, J. E. Characterizing Bacterial Volatiles using Secondary Electrospray Ionization Mass Spectrometry (SESI-MS). J. Vis. Exp. (52), e2664, doi:10.3791/2664 (2011).
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.
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 μm filter (5), as an additional measure of protection, and vented to a chemical hood. Inset: The SESI electrospray needle is a silica capillary (40 μm 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 °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 μm 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 – 500 Da, MCA mode, 40 scans, 3 s/scan, and 2 min total analysis time.
1. Culturing system
2. Biological experiment: set-up and safety considerations
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.
4. Turning on and tuning the SESI-MS for analysis
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.
5. Obtaining a volatile fingerprint of your bacterial culture
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 °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.
Figure 2. Blank-subtracted positive ion mode SESI-MS spectra (20 – 150 m/z) of E. coli K12 and P. aeruginosa PAO1 volatiles after 24 h aerobic growth in LB-Lennox at 37 °C. For more details about the peaks observed in the SESI spectra, please refer to Zhu, et al. 8.
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
No conflicts of interest declared.
This work is funded by NIH grant P20 RR021905-01, CF RPD grant STANTO07R0, and NASA grant NNH09ZNE002C.
|API-3000 Triple Quadrupole||Instrument||SCIEX||Purchased with Analyst 1.4.2 (Applied Biosystems)|
|SESI Ion Source||Instrument||Custom-made; See reference 6|
|Gas flow meter||Equipment||Cole-Parmer||EW-03217-74|
|Carbon dioxide||Equipment||Airgas||CD I300||â€°Â¥ 99.99% pure|
|Nitrogen||Equipment||Airgas||NI UHP300||Ultra high purity|
|100 mL glass media bottles||Equipment||VWR international||89012-114||GL45 screw threads|
|Bottle caps with luer ports||Equipment||Bio Chem Fluidics||00945T-3||Cap assembly|
|Luer port plugs||Equipment||Bio Chem Fluidics||009LP||Cap assembly|
|Tubing 1/4" (OD) x 1/8" (ID)||Equipment||Cole-Parmer||EW-95875-02||Cap assembly & gas transfer lines|
|Tubing 1/8" (OD) x 1/16" (ID)||Equipment||Cole-Parmer||EW-06605-27||Cap assembly|
|Two-way valves||Equipment||Cole-Parmer||07391-04||Cap assembly|
|Filter, Grade AAQ||Equipment||Balston Filters||9922-05|
|Formic acid, LC/MS grade||Reagent||Fisher Scientific||A117-05AMP||Electrospray solution|
|Methanol, LC/MS grade||Reagent||Fisher Scientific||A456-500||Electrospray solution|
|Water, LC/MS grade||Reagent||Fisher Scientific||W6-500||Electrospray solution|