Instantaneous Response of Bacteria to External Stimuli Monitored by

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Instantaneous Response of Bacteria to External Stimuli Monitored by Syringe Spray Mass Spectrometry Lili Hu, Chuangui Zhou, Hang Li, Mei Zhang, and Wei Xu Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b02443 • Publication Date (Web): 31 Aug 2018 Downloaded from http://pubs.acs.org on September 1, 2018

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Analytical Chemistry

Instantaneous Response of Bacteria to External Stimuli Monitored by Syringe Spray Mass Spectrometry

Lili Hu,1 Chuangui Zhou,1 Hang Li,1 Mei Zhang2 and Wei Xu1*

1 School of Life Science, Beijing Institute of Technology, No. 5 South Zhongguancun Street, Haidian Dist, Beijing, China 2 School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing, China

*Email: [email protected]

Conflict of interest The authors declare that they have no conflict of interest.

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Abstract Microbial adaptation to environmental stress involves complex adaptations of bacteria. Many such responses are transient and dynamic. However, monitoring the dynamic responses of live bacteria to stimulations at the molecular level remain a challenge. This work describes the development of syringe spray mass spectrometry (MS) method that allows direct analyses of molecules released by the bacteria in responses to external stimuli with second level time resolution. We report the application of this technique to visualize the dynamic release of small molecules from Escherichia coli (E. coli) under ethanol and isopropanol treatments. With the unique time-resolved capability, detailed destruction process of alcohol on bacteria cell wall could be observed. Compared to other ethanol concentrations, 75% ethanol showed stronger damages to lipopolysaccharide (LPS) and peptidoglycan located on E. coli cell wall. Furthermore, isopropanol showed stronger liposolubility and permeability, and an equilibrium could be achieved in a much shorter time.

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Analytical Chemistry

Introduction: With the explosive interest in microbiome effects on human health and the emergence of super bacteria, there are increasing demands for rapid and powerful microorganism analysis techniques.

1-6

Capturing the dynamic responses of

microorganism to environmental changes and stimulus is important for understanding microbial adaption and evolution, which have practical significance in antibiotic drug development and microbiomics study.

7-13

Conventional biochemical tests of

microorganisms are time consuming, and most techniques are mainly used to study the static states of microorganisms.

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Due to its high specificity and sensitivity,

mass spectrometry is a powerful analytical technique in biochemical analyses, which is widely used in microbiology.

18-21

Matrix-assisted laser desorption ionization

time-of-flight (MALDI-TOF) MS is currently the standard method for bacterial identification.

22,23

MS imaging is a tool allowing the mapping of biomolecular

distributions in biological tissues.24-27 MS imaging methods, such as desorption electrospray ionization (DESI),28 nanoDESI,29,30 MALDI,31 secondary ion MS (SIMS)32 and laser ablation electrospray ionization (LAESI) imaging,33 have also been applied in the studies of microbe-drug interaction, microbial metabolism and communication. Syringe spray was previously developed in our group as an integrated and disposable device for the direct analysis of toxic agent in water.

34

In this study,

syringe spray was coupled with MS and extended for the analyses of solid phase samples, as well as monitoring the dynamic responses of live bacteria under irritations or stimulations. Syringe spray was first optimized and demonstrated for the direct analyses of powder, soil and plant tissue samples. After optimization, it was applied to monitor the dynamic responses of live bacteria under external stimulations. In previous bacteria studies, MS imaging techniques were mainly applied in the spatial mapping of microbial colonies. To map a relatively large area with high spatial resolution, it would normally take several hours to finish a MS mapping experiment. 2

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On the other hand, syringe spray MS does not have the capability of spatial mapping, but it could achieve second level time responses of a group of bacteria. As a proof-of-concept demonstration, the well-known alcohol disinfection effects were investigated in a dynamic fashion and at the molecular level. Ethanol and isopropanol were used to stimulate E. coli, respectively. The instantaneous responses of E. coli were characterized by monitoring biomolecules eluted from E. coli over time. Alcohol could effectively destruct E. coli cell wall and cause protein denaturation. However, different alcohols at different concentrations showed distinct disinfection processes in the time domain. The use of syringe spray for quick bacteria differentiation was also described in this work.

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Analytical Chemistry

Experimental Section Bacteria and culture conditions. Bacteria were kept as glycerin stored prepared in Luria Broth (LB) medium containing 50% glycerol at −80 °C. The cultures were aliquoted from the stock tubes, then thawed at room temperature (specify as in 24 °C). Bacteria solution was incubated in conical flask with 30 ml LB medium (vol/vol, 1%) in an orbital shaker (ZHWY-2102, Shanghai Zhicheng Analytical Instrument Manufacturing Co. LTD, ShangHai) at 37 °C and 180 rpm. After 12 h incubation, bacteria solution (1 mL) was transferred into a 1.5 mL centrifuge tube. Bacteria solution was then centrifuged for 1 min at 6000 rpm (centrifuge 5424, Eppendorf, Germany), and the supernatant was preserved. Add distilled water to the suspension and centrifuge at the same condition for three times. To load bacteria onto the filter membrane, bacteria were first transferred from the centrifuge tube into a disposable syringe. A filter was mounted in front of the syringe. By pushing the plunger, bacteria could be loaded onto the membrane. Take a new syringe filled with a certain amount of irritant in solution and install the filter loaded with bacteria. A modified needle is then mounted on the front end of the filter. The whole device was then fixed to a peristaltic pump. Angle and relative position of the needle relative to the mass spectrometer inlet could be adjusted as described in the optimization section. After adjusting flow rate of the peristaltic pump (6 – 15 µL/min), MS analysis could now be carried out. Figure S4 shows that washing and treatment procedures didn’t inactivate these bacteria. In control experiments, nanoESI MS was performed to analyze bacteria supernatant. To obtain bacteria supernatant, bacteria was washed twice by distilled water. After that, bacteria were crushed by the ultrasonic cell crusher (VCX 800, Sonics & Materials, INC.). Then the solution was centrifuged at a speed of 8000 rpm. The supernatant was used for control experiments.

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Material and sample.

All experiments were carried out using a Bruker HCT ion

trap mass spectrometer (Bruker Daltonics Inc., MA, Germany). Nitrogen was used as the drying gas (flow rate, 10 L/min; temperature, 150 °C). The Ultra scan mode was applied, and the mass range was set from 100 to 1000 Th in the positive ion mode. Peptide Met-Arg-Phe-Ala (MRFA), methanol, progesterone and atrazine were purchased from Sigma-Aldrich (St. Louis, MO). Deionized water was purchased from Wahaha Company (Hangzhou, China). Sample solutions were prepared individually. Amoxicillin capsules were purchased from Lukang Pharmaceutical co., Inc. (Shandong, China). DMMP was purchased from Beijing chemical reagent co., LTD (Beijing, China). Allicin was extracted from the menthol solution of fresh garlic slice. Isopropyl alcohol and ethanol were purchased from Xilong Scientific Co., Ltd.; Soil samples were gathered in the campus of Beijing Institute of Technology. Sewage water was collected from the mop sewage bucket in our laboratory. The micro syringe pump (model KDS-100) was purchased from KD Scientific Inc. (Holliston, MA). The disposable sterile syringe was purchased from Zhiyu Medical Equipment Inc. (Jiangsu, China). The membrane was purchased from Jinteng Experimental Equipment Co. (Tianjin, China). A glass capillary with a tip diameter of ~5 µm was prepared by pulling a glass capillary (0.8 mm i.d.; 1.5 mm o.d.) with a micropipette puller (model P-1000, Sutter Instrument Inc., USA), which was used as the nano-electrospray ionization (nano-ESI) source for comparison study. E. coli, Pseudomonas aeruginosa, Klebsiella pneumonia, Baumann's acinetobacter, Staphylococcus aureus used for classification identification and E. coli (DH5α) used for dynamic response monitoring were provided by National Institute for Communicable Disease Control and Prevention, Chinese Center for Disease Control and Preventio.

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Analytical Chemistry

Methods: In a syringe spray MS experiment as shown in Figure 1, a chemical inert enameled wire was inserted into the needle to enable an effective electrospray. Other materials, such as bamboo and cotton wires, were also tested, but they are not used in later experiments due to a strong chemical background problem (please refer to Supporting Information for details). A PES (polyethersulfone) membrane filter was mounted between the syringe barrel and the stainless-steel needle. After optimization (Figure S2), the syringe needle was placed ~2.5 mm away from the MS inlet for MS analysis, and an angle (30°) was kept between the needle and the MS inlet. A micro syringe pump was used to inject solvents from the syringe through the PES membrane filter to the needle. After applying a high voltage between the needle and the MS inlet, a Taylor cone was formed (Figure 1) to effectively generate ions. With the PES membrane (pore size 0.22 µm; 13 mm o.d.) assisted in filtering larger particles from interfering MS analysis, in situ sampling and real time MS analysis could be performed using this syringe spray device. The capability of analyzing samples in complex matrices, such as direct analyses of effective ingredient in a drug capsule, herbicide residue in soil samples and bio-ingredients in a garlic tissue were first demonstrated. First, the medical drug, amoxicillin powder in a capsule could be loaded into the syringe. After adding methanol and shaking for multiple times, amoxicillin (365 Da) could be directly ionized and observed in the mass spectrum. As shown in Figure 2a, a limit of detection of 1 µg/mL was achieved. Other ingredient in the amoxicillin powder, such as starch could be effectively filtered, preventing the blocking and contamination of the electrospray tip. In environmental applications, there are also a lot cases in which solid samples need to be analyzed, such as the analysis of pesticide residues in soil. 35 To prepare the simulated pesticide polluted soil samples, 10 mg atrazine (215 Da) in water solution was mixed with 10 g soil sample. After fully mixed, the mixture was solarized to volatize water. Then we took 1 mg of this solid soil sample and further diluted with methanol to targeted 6

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concentration. This methanol diluted soil samples could be directly loaded in the syringe and performing MS analysis. Figure 2 b plots the linear range of detection curves of atrazine in soil, and a limit of detection of 1 µg/mL was obtained. As shown in Figure 2b insets, the photos before and after membrane filtering suggesting that it is capable of handling samples with complex matrices. Insoluble solid samples could also be processed and analyzed by the syringe spray device, and the biochemical ingredients could be examined directly without sample pretreatments. As shown in the next experiment, a garlic

36,37

slice (~0.1 g, 1 mm thick) was directly loaded into the

syringe. Then, ~1 mL solvent (1:1 water methanol by volume) was filled into the syringe. By shaking about 10 seconds, biochemical molecules could be extracted and analyzed by syringe spray. As shown in Figure 2c, many biochemical constituents in the garlic tissue could be identified from the mass spectrum, including amino acids, allicin, alliin and polysaccharides. Figure 2c also shows some representative tandem mass spectra of these chemicals, including protonated arginine and polysaccharide (DP = 6, DP = 8), as well as allicin cations [M+K]+. The tandem mass spectra of lysine, leucine, alliin were also plotted and shown in the Figure S3. Since only portions of the soluble chemicals at the garlic slice surfaces were extracted and dissolved in the solvent, quantitative analyses are difficult to be carried out using the current method without further modifications.

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Analytical Chemistry

Results: In terms of bacteria analyses, we first tested the responses of E. coli to different concentrations of ethanol, and whole live bacteria were first loaded onto the PES membrane directly after rinsing (refer to Supporting Information for details). Ethanol was spiked into the solution, which was then loaded into the syringe. E. coli attached on the membrane will be stimulated when this solution stream was driving through the membrane. Metabolites or stress reaction efflux dissolved in the solution could then be ionized and analyzed by the mass spectrometer. The collected mass spectrum changes with respect to time, and different mass peaks show up at different time points (Figure 3). Figure3a shows three typical mass spectra collected using the syringe spray at different time points, and 75% ethanol in water was used as the stimuli, as well as the electrospray solution. The collected mass spectrum pattern changes with respect to time, and different mass peaks appeared in the mass spectra. To validate feasibility of syringe spray, the mass spectra collected from syringe spray were compared with that collected from the conventional nano-ESI method. In contrast to the syringe spray, a nano-ESI source does not have the capability of handling whole bacteria directly. Therefore, E. coli was first extracted from the culture medium and then soaked with 75% ethanol solution 30 min. After centrifugation, the supernatant was loaded in a nano-ESI source and MS analyzed. ( Figure S5 shows the E. coli was treated with ethanol solutions of different concentrations and the supernatant was directly extracted and nanoESI analyzed). The bottom figure in Figure 3b plots the corresponding mass spectrum collected using a nano-ESI. The top figure in Figure 3b plots the averaged mass spectrum collected using the syringe spray over the whole analysis time (~30 mins). It is interesting to find that some mass peaks were significantly enhanced with syringe spray. For instance, ions at m/z 222.2, 257.4, 294.3 and 742.5 Th could be observed in both spectra. Results suggested that syringe spray could effectively ionize small molecules presented in E. coli. However, besides the m/z domain information, syringe spray 8

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could provide information in another dimension, the time-domain response. For example, the relative intensities of ions at m/z 222.2, 257.4, 294.3 and 742.5 Th would change with respect to time. After performing tandem MS, the mass peaks at m/z 222.2, 294.3, 257.4 and 742.5 Th were identified as N-acetylglucosamine (NAG), N-acetylmuramic acid (NAM), palmitic acid and phosphatidylethanolamine (PE(16:0/17:1)), respectively. Total ion chromatogram (TIC) and extracted ion chromatogram (EIC) of NAG, NAM, palmitic acid and PE were plotted and compared at different ethanol concentrations (Figure 4a-d). Figure 4e plots the EIC peak area of these four biomolecules, and this peak area characterizes the total amount of biomolecules released from E. coli during ethanol treatment. Figure 4f plots the EIC peak starting and ending time at half maximum, which could be used to characterize the response speed and duration of E. coli to stimuli. Repeated experiments were also carried out with 75% ethanol concentration ( Figure S6 ), indicating reasonable repeatability of the current method. Furthermore, there are two major factors that would affect the mass spectra when using various ethanol concentrations as the solvent. First, different ethanol concentrations would dissolve or damage the cellular wall differently, which is actually one of the phenomena that this manuscript is trying to characterize. Second, different ethanol concentrations would affect the electrospray ionization efficiency. The current study is based on the assumption that ethanol concentrations would have little effects on the ionization efficiency of target compounds. Figure S7 in the Supporting Information shows that similar ionization efficiency was obtained for rhodamine B at different ethanol concentrations. However, since bacteria is a complex system and untargeted analysis is performed, it cannot rule out the possibility that the four identified compounds are affected by different ethanol concentrations. It should also be noticed that no internal standard was used in the current study. The relative quantitation was based on absolute ion intensities.

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Analytical Chemistry

There are two possible sources for NAG: first from the peptidoglycan located inside the outer membrane; second from the LPS located at the outside wall of E. coli. Peptidoglycan are composed of peptides and polysaccharides, and NAG is one of the residues on polysaccharide. Lipid A is the toxic component of LPS, which consists of two NAGs and five saturated fatty acid chains. Palmitic acid belongs to one of these five fatty acids, so palmitic acid is part of LPS. NAM links the short peptide chain with NAG in the peptidoglycan. Peptidoglycan is believed to be the source of NAM. PE actually counts for ~80% of total phospholipids in E. coli. The PE observed in this experiment is suspected to be from the inner surface of cell wall or from parts inside the cell wall. The detection of both palmitic acid and NAM in all cases indicated that both LPS at the outer cell wall and peptidoglycan at the inner cell wall were eluted. Results also suggested that NAG comes from both LPS and peptidoglycan. However, ion intensity variations of NAM, NAG, palmitic acid and PE have different release patterns when different ethanol concentrations were used. A strong and quick PE release was only observed with the 75% ethanol, indicating that 75% ethanol could effectively damage the cell wall and cause the release of PE from inner surface of E. coli cell wall or even from the cytoplasmic cell membrane. Since PE has important roles in the structure and assembly of membrane proteins, which could then cause E. coli metabolic disorders, such as the transportation of lactose, phenylalanine and γ-aminobutyric acid. Therefore, a significant loss of PE would cause fatal damage to E. coli. Transmission electron microscope images confirm that ethanol could cause the coagulation of cytoplasmic proteins under our experimental conditions (Figure S8). Treated with 100% ethanol, a loss of PE was also observed, but a slower and relatively stable PE signal was obtained from the mass spectrometer. Lower PE signals were measured when using 40% and 50% ethanol solutions. This is likely due to the hydrophobic group of ethanol destroyed the hydrophobic interactions (such as the Van der Waal and electrostatic forces) within a biomolecule, however water 10

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molecule could relax the attractions between hydrophilic groups within a biomolecule, such as the hydrogen bonds. The Van der Waal force, electrostatic force, hydrogen bonds are critical forces to maintain the steric structures of proteins, destruction of these forces by the combination of ethanol and water molecules would denature proteins in bacteria and cause bacteria disinfection. Experiments were also carried out using isopropanol to stimulate five different types of foodborne bacteria, including four common gram-negative bacilli and a gram-positive coccus. Four gram-negative bacilli, E. coli, Pseudomonas aeruginosa, Klebsiella

pneumonia,

Baumann's

acinetobacter

and

the

gram-positive

Staphylococcus aureus were collected from the excreta and secretions of patients. We found that these bacteria have much faster responses to isopropanol, and equilibrium could be achieved within several minutes. As an example, Figure 5a shows the dynamic response of E. coli to 50% isopropanol in water solution, and small variation in the mass spectrum are observed after 3 mins. This may be due to the fact that isopropanol has two methyl group, which has stronger liposolubility and permeability. Furthermore, since ethanol has stronger polarity than isopropanol, less NAG but more PE were observed in Figure 5a than those in Figure 3. Besides monitoring dynamic responses of bacteria, syringe spray MS could also be used to identify and distinguish bacteria species. As a proof-of-concept demonstration, these five bacteria were analyzed, and five strains of each bacterium were sampled and underwent syringe spray MS analyses. Mass spectra of the other four bacteria after equilibrium were also shown in Figure 5b. To perform the multivariate statistical analyses, list of all m/z ratios and their relative intensities of a mass spectrum were imported into MATLAB (MathWorks, Inc., Natick, MA), in which the normalization and de-noising operations were performed. Then the processed data was imported into SIMCA-P 14.1 (Umetrics AB, Umea, Sweden). Results in Figure 5c show that these five bacteria could be well separated after applying the supervised Orthogonal Partial Least Squares Discriminant Analysis 11

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Analytical Chemistry

(OPLS-DA). The number of significant components was computed by the default rules within SIMCA, and a seven-round of k-fold cross-validation was used in the auto-fitting prediction. Results show that there are big differences between gram-positive and gram-negative bacteria, and they could be first well separated in the PC1 dimension. As shown in Figure 5b for gram-positive bacteria (Staphylococcus aureus), there are less number of mass peaks at the high mass range (m/z 600-800 Th), which correspond mainly to lipid ions. This is due to the fact that gram-positive bacteria have much thicker cell walls mainly composed of peptidoglycan, which prevents the damage and leakage of lipid molecules from the inner cytoplasma membrane. In the current study, five bacteria species could be well separated using the mass spectra after equilibrium. In the future, it is possible to utilize the time-resolved capability of this method to better differentiate either large number of bacteria species or identify species with very close characteristic mass spectra.

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Conclusions: With the development of syringe spray MS, we have demonstrated that it is possible to obtain the dynamic response of live bacteria to external stimuli at the molecular level. Our results showed that E. coli have different response curves to different stimuli, which provide us new insights into the mechanism of alcohol sterilization and a method to evaluate sterilization effectiveness. Based on the fast scan speed of a MS instrument, second level time domain resolution could be achieved, and rich molecular information associated with bacteria degeneration is available through MS and tandem MS analyses. Although the method is capable to achieve sub-second level time resolution, minute-level bacteria responses are observed to alcohol stimuli. This ability to capture time resolved molecular information of live bacteria has the potential to be used for the in-depth investigation of bacteria metabolism, bacteria-drug and bacteria-bacteria interactions.

Acknowledgments This work was supported by National Key research and development plan (2018YFF0212500), NNSF China (21827810, 21475010, 61635003) and BNSF (16L00065).

Supporting information is available at the journal’s website.

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Analytical Chemistry

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(22) Singhal, R.; Carrigan, J. B.; Wei, W.; Taniere, P.; Hejmadi, R. K.; Forde, C.; Ludwig, C.; Bunch, J.; Griffiths, R. L.; Johnson, P. J.; Tucker, O.; Alderson, D.; Günther, U. L.; Ward, D. G. J. Proteomics 2013, 80, 207-215. (23) Si, T.; Li, B.; Comi, T. J.; Wu, Y.; Hu, P.; Wu, Y.; Min, Y.; Mitchell, D. A.; Zhao, H.; Sweedler, J. V. J. Am. Chem. Soc. 2017, 139, 12466-12473. (24) Mainini, V.; Lalowski, M.; Gotsopoulos, A.; Bitsika, V.; Baumann, M.; Magni, F. In Clinical Proteomics: Methods and Protocols, Vlahou, A.; Makridakis, M., Eds.; Springer New York: New York, NY, 2015, pp 139-164. (25) Aichler, M.; Kunzke, T.; Buck, A.; Sun, N.; Ackermann, M.; Jonigk, D.; Gaumann, A.; Walch, A. Lab. Invest. 2018, 98, 141-149. (26) Scott, A. J.; Jones, J. W.; Orschell, C. M.; MacVittie, T. J.; Kane, M. A.; Ernst, R. K. Health Phys. 2014, 106, 120-128. (27) Zavalin, A.; Yang, J.; Hayden, K.; Vestal, M.; Caprioli, R. M. Anal. Bioanal. Chem. 2015, 407, 2337-2342. (28) Claude, E.; Jones, E. A.; Pringle, S. D. In Imaging Mass Spectrometry : Methods and Protocols, Cole, L. M., Ed.; Springer New York: New York, NY, 2017, pp 65-75. (29) Lanekoff, I.; Burnum-Johnson, K.; Thomas, M.; Short, J.; Carson, J. P.; Cha, J.; Dey, S. K.; Yang, P.; Conaway, M. C. P.; Laskin, J. Anal. Chem. 2013, 85, 10.1021/ac401760s. (30) Watrous, J.; Roach, P.; Heath, B.; Alexandrov, T.; Laskin, J.; Dorrestein, P. C. Anal. Chem. 2013, 85, 10385-10391. (31) Bloom, A.; Winograd, N. Surf. Interface Anal. 2014, 46, 177-180. (32) Urbanek, A.; Hölzer, S.; Knop, K.; Schubert, U. S.; von Eggeling, F. Anal. Bioanal. Chem. 2016, 408, 3769-3781. (33) Zou, J.; Talbot, F.; Tata, A.; Ermini, L.; Franjic, K.; Ventura, M.; Zheng, J.; Ginsberg, H.; Post, M.; Ifa, D. R.; Jaffray, D.; Miller, R. J. D.; Zarrine-Afsar, A. Anal. Chem. 2015, 87, 12071-12079. (34) Si, X.; Hu, L.; Xu, W.; Li, H.; Li, C. Int. J. Mass spectrom. 2017, 423, 15-19. (35) Wang, P.; Rashid, M.; Liu, J.; Hu, M.; Zhong, G. Food Chem. 2016, 212, 420-426. (36) Zhang, H.; Chingin, K.; Zhu, L.; Chen, H. Anal. Chem. 2015, 87, 2878-2883. (37) Kim, S.; Park, S.-L.; Lee, S.; Lee, S.-Y.; Ko, S.; Yoo, M. Food Chem. 2016, 211, 555-559.

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Analytical Chemistry

Figure 1

Figure 1. Schematic diagrams of (a) syringe spray setup and (b) monitoring the dynamic process using syringe spray MS.

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Figure 2

Figure 2. The linear detection range of amoxicillin directly extracted from drug capsule (a) and atrazine in soil (b); (c) analysis of a garlic slice directly by syringe spray, mass spectrum and the corresponding tandem mass spectra of arginine, allicin and saccharide in the garlic slice tissue.

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Analytical Chemistry

Figure 3

Figure 3. (a) Time resolved syringe spray mass spectra of E. coli under ethanol stimulation; (b) top: the averaged syringe spray mass spectrum over the whole analysis time; bottom: the corresponding mass spectrum collected using conventional nanoESI. (c) MS/MS of N-acetylglucosamine (m/z 222.2), Palmitic acid (m/z 257.4), N-acetylmuramic

acid

(m/z

294.3)

and

(PE[(16:0/17:1)+K]) (m/z 742.5).

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Phosphatidylethanolamine

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Figure 4

Figure 4. The dynamic responses of E. coli under the stimulation of solvents with different ethanol concentrations: (a) 40%, (b) 50%, (c) 75% and 100%, and the TIC and EICs of four ions (NAG, NAM, palmitic acid and PE) were monitored. EIC peak areas (e), EIC starting and ending times (f) of these four ions.

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Analytical Chemistry

Figure 5

Figure 5. (a) The dynamic response of E. coli to 50% isopropanol in water solution. (b) Typical mass spectrum of four other bacteria: Staphylococcus aureus, Baumann’s Acinetobacter, Klebsiella pneumonia and Pseudomonas aeruginosa. (c) OPLS-DA analysis of these five bacteria.

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