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Quantitative Analysis of Metabolites at the Single-cell Level by Hydrogen Flame Desorption Ionization Mass Spectrometry Junbo Zhao, Fang Zhang, and Yinlong Guo Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b04422 • Publication Date (Web): 15 Jan 2019 Downloaded from http://pubs.acs.org on January 16, 2019

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Analytical Chemistry Title: Quantitative Analysis of Metabolites at the Single-cell Level by Hydrogen Flame Desorption Ionization Mass Spectrometry Jun-Bo Zhao, Fang Zhang, and Yin-Long Guo* State Key Laboratory of Organometallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China *Corresponding author: Prof. Yin-Long Guo State Key Laboratory of Organometallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai Shanghai Institute of Organic Chemistry, CAS, 345 Lingling Road, 200032, Shanghai, China Email: [email protected] Phone: +86 25 54925300 Fax: +86 25 54925314

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Quantitative Analysis of Metabolites at the Single-cell Level by Hydrogen Flame Desorption Ionization Mass Spectrometry Jun-Bo Zhao, Fang Zhang, and Yin-Long Guo* State Key Laboratory of Organometallic Chemistry and National Center for Organic Mass Spectrometry in Shanghai, Center for Excellence in Molecular Synthesis, Shanghai Institute of Organic Chemistry, University of Chinese Academy of Sciences, Chinese Academy of Sciences, 345 Lingling Road, Shanghai, 200032, China ABSTRACT: To date, direct quantitation of cellular metabolites at the pico-liter level or in a single cell is still a challenge due to tiny sampling materials, the accuracy of the sampling volume, and the ubiquitous matrix effect. Herein, pico-liter magnitude quantitative analysis was performed using a pressure-assisted micro-sampling probe coupled to the hydrogen flame desorption ionization mass spectrometry (HFDI-MS). The sampling was accurately controlled with a pico-liter pump and the analytes were rapidly vaporized and quantitatively transferred to the gas phase by adequate heat. The vapor-phase analytes reacted with protonated water cluster ions by the proton-transfer reaction (PTR). The accurate sampling, flash thermal desorption and proton-transfer ionization processes were conducted spatiotemporally, which could greatly reduce matrix effects to facilitate the quantitation of analytes without the internal standard. Furthermore, this workflow enabled the quantitation of cellular metabolites at pico-liter/single-cell level.

Cell is the basic unit of life that is responsible for the maintenance and expression of genetic materials, providing a location for anabolism, metabolism, etc. To better study the composition, distribution and content variation of metabolites in a single cell, it is necessary to characterize and quantify the ingredients and metabolites at the single-cell level, which gives us significant insights in the studies of life science.1,2 Mass spectrometry (MS) analysis provides unique insights into the metabolite diversity and cell heterogeneity due to its outstanding advantages of high sensitivity, high specificity, and wide molecular coverage, but the quantitative analysis of metabolites at the single-cell level is still a challenge.3-8 Secondary ion mass spectrometry (SIMS) and matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) have proven the ability to perform quantitative analysis in single cells for its metabolites composition. Ewing's group successfully developed SIMS for the relative quantitation of cholesterol and phospholipid in single-cell membranes by incubating cells with a deuterated standard prior to analysis.9,10 Sweedler's team achieved the relative and absolute quantitation of signaling peptides in a single cell by adding an internal standard to the matrix solution.11 These vacuum-based ion sources have been shown to provide excellent sensitivity and high throughput for the single-cell analysis, but the analyzed cells were far from their natural state due to the loss of water and sometimes were affected by the matrix in the low m/z range. In this decade, ambient MS methods have demonstrated their potential for rapid, direct, in situ and in vivo qualitative and quantitative analysis in a single-cell system.12 Masujima's group made great efforts in developing a live single-cell MS, which employed a metal-coated nanospray tip to extract metabolites from single mammalian and plant cells under a video microscope followed by nano-electrospray ionization MS (nano-ESI-MS).13-17 This approach was applied to the

identification and quantitation of plant hormones in single plant cells by adding isotope-labeled standards into the ionization solvent.18 In addition, to accurately suck the volume of the samples, they developed live single-cell MS combined with three-dimensional holographic and tomographic laser microscopy for the spatial evaluation and quantitative analysis of a HepG2 cell.19 Initially, Hiaroka's group proposed probe electrospray ionization MS (PESI-MS) for the analysis of liquid samples under ambient and open-air conditions, and this method provided guidance and feasibility for single-cell analysis.20 Then, Zhang's group first demonstrated qualitative and quantitative analysis with PESI-MS for the detection of metabolites at the cellular and subcellular levels. The quantitative analysis of sucrose in Allium cepa bulb was achieved using this technique by adding an appropriate internal standard to the assistant solvent.21 To enhance the coverage of biomolecules and minimize the isomer interferences in single-cell analysis, Vertes's group demonstrated the combination of capillary micro-sampling with ESI-MS and ion mobility separation (IMS) for the identification of plentiful metabolites in single human and plant cells.22,23 This method showed high potential and advantages for the analysis of complex metabolites in a single cell. Moreover, laser ablation electrospray ionization MS was first demonstrated for in situ analysis of metabolic variations in plant cells by the Vertes group.24-26 To extract the analytes from a single cell for mass spectrometric analysis under ambient conditions, Verbeck's group described a nanomanipulation-coupled nanospray MS for examining the lipid content of individual cells and profiling of triglycerols in healthy and tumorous tissues, and lipid droplets.27,28 These techniques shared similarities such as the necessary introduction of internal standards to compensate for matrix effects, but the results sometimes did not reflect the true content of the metabolites in a single cell due to the uncertainty of the sampling volume.


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Analytical Chemistry

Figure 1. Schematic diagram of the experimental setup and analysis process. (a) Two kinds of holly leaves: old-growth leaf and tender leaf. (b) Direct sampling from plant single cells in a microscope. (c) Microscope image of the probe sampling and post-sampling. (d) Schematic of hydrogen flame desorption ionization source. (e) The EIC spectrum of the arginine was obtained at 2 mM in SRM mode (m/z 175→116) by HFDI-MS.

To control and measure the sucked volume in a single cell with accuracy, pressure-assisted micro-sampling technique was developed. Nonami's group was the first to introduce the concept of a cell pressure probe for MS-based metabolite profiling in single plant cells.29,30 The capillary filled with an oil mixture was connected to a pressure transducer; thus, the volume of the cell sap sucked in the capillary tip could be accurately controlled and measured. It was notable that this technique could suck the deep cell sap with minimal contamination from other surrounding cells and provide an important prerequisite (the exact sample volume) and orientation for the quantitative analysis of metabolites in single cells. The metabolite characterization and relative quantitation could be readily realized by adding known volumes of isotope-labeled standards to the cell sap samples inside the capillary tip.29 In addition, Sweedler's group developed highly sensitive capillary electrophoresis ESI-MS (CE-ESI-MS) for the characterization of cell heterogeneity and quantitative analysis of metabolites from a single neuron.31-33 Then, Nemes's group described a microprobe single-cell CE-ESI-MS for the analysis of identified cells in the live frog embryo.34 This technique significantly enhanced detection sensitivity and quantitative repeatability compared to whole-cell dissection by minimizing chemical interferences and ion suppression effects from the culture media. Moreover, Baker's group demonstrated pressure-assisted nanopipettes for reproducibly collecting fluids from single Allium cepa cells and brain tissue section of a mouse.35 The collected fluids were subsequently subjected to MALDI-MS analysis. The advantage of these techniques was that the localization of the tip into the target cell was conducted under a microscope and that the sucked volume of sample was controlled and measured. However, these ionization methods mentioned above for the quantitative analysis of analytes in single-cell analysis often required internal standards to compensate or reduce the effects of matrix and then elucidated the relative

abundance of the examined metabolites. At the same time, the limited availability of deuterated standards might also be a concern. To achieve the direct quantitative analysis of metabolites at a single-cell level, herein, a pressure-assisted micro-sampling probe coupled with HFDI-MS was introduced. First, a pressure-assisted micro-sampling GlassTips probe of 2.5±0.5 μm in diameter as the sample collection device was used to suck analytes in the cell or sample solutions. The sucked volumes were controlled via a pico-liter pump by adjusting the gas pressure and the duration time. The operation was conducted under a microscope, and the volume was calculated from the observed diameter and height of the stable meniscus in the tip (see Figure S-1). Then the reproducibility and stability of the signal intensity (peak area) was improved by accurately controlling the probe tip sampling volume. Second, the hydrogen flame provided a small heating zone for the fast desorption of the analytes. Then the vapor-phase analytes entered into a flame plasma plume and reacted with protonated water cluster ions to form analyte ions at atmospheric pressure. This plasma-based ambient desorption ionization process was similar to atmospheric pressure chemical ionization, in which the vaporization of the analytes and PTR were separated in both time and space.36-40 Thus, the matrix effects (ion suppression) of this method were typically considered to be far less severe than the spray-based ambient desorption ionization method. Moreover, analysis of small volume samples at the single-cell level could also minimize or reduce the matrix effect due to the lower amount of salts or other interfering ions.34 This method was used for direct quantitation of molecules in complex matrices without adding internal standards, and the signal intensity obtained could truly reflect the concentration of the analytes in complex samples.

EXPERIMENTAL SECTION



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Figure 2. (a) Signal intensity versus the flame height (desorption temperature). (b) Signal intensity versus the distance between the probe tip and the MS inlet. (c) Flame temperature versus flame height. (d) A calibration curve showed the linear correlation of the peak area of lysine in the volume range of 10 to 500 pL. The signal intensity (peak area) was obtained by monitoring the protonated lysine (1 mM, aqueous solution) in SIM scan mode three times.

Chemicals and Materials. All aqueous solutions, if not otherwise noted, were prepared with deionized water (purified by a Milli-Q plus apparatus; Millipore, Molsheim, France). HPLC-grade ethanol was purchased from Merck (Darmstadt, Germany). Lysine, arginine, aspartic acid and cinene were purchased from Aladdin (Shanghai, China). Quercetin, methyl salicylate, sinensetin (SIN, 3,4,5,6,7-pentamethoxyflavone) and phthalic acid were purchased from J&K (Shanghai, China). Tangeretin (TAN, 4,5,6,7,8-pentamethoxyflavone) was purchased from Titan (Shanghai, China). Melamine and 4-methylstyrene were purchased from TCI (Tokyo, Japan). Holly leaves were collected from holly plants on both sides of the garden road in Shanghai. Purple Allium cepa bulbs and oranges were purchased from a local supermarket in Shanghai. The test materials were all stored at 4 °C before analysis. The cross-section cells of holly leaves, the inner epidermal cells of Allium cepa, and the orange peels were used for the quantitation experiments. The GlassTips probes (1.2 mm OD, 0.69 mm ID, 2.5±0.5 μm tip, purchased from New Objective, Inc.) were made using the highest-grade borosilicate glass. Hydrogen gas was pre-mixed with the auxiliary gas of carbon dioxide in a gasbag at a volume ratio of 1 to 1. A stainless steel tube with an inner diameter of 0.8 mm was used as the gas flow tube, and the outlet was lit to produce a hydrogen flame. Preparation of Samples. A methyl salicylate stock solution (100 mM) was prepared in ethanol solution, and an arginine stock solution (20 mM) was prepared in methanol/water (1:1, v/v) solution. Those solutions were respectively diluted to a series of working solutions of 0.1, 1.0, 2.0, 5.0 and 10 mM. The 2 mM methyl salicylate solution was tested for reproducibility. All solutions were stored at 4 °C and returned to room temperature before use.

A piece of holly leaf (380.8 mg, 3×4 cm2) was put into a clean glass mortar and ground for 5 min. After that, 150 μL of ethanol/water (1:1, v/v) was added, and the mixture was ultrasonicated for 5 min. Finally, the suspension was transferred to a 1.5-mL microtube and centrifuged at 12,000 rpm for 5 min. The supernatant was collected for testing. The spiked solutions with 0.1, 1.0, 2.0, 5.0 and 10 mM methyl salicylate were prepared by dissolving the compound in the supernatant. A few pieces of inner epidermal cell layer (210.8 mg) peeled from an Allium cepa sample were put into a clean glass mortar and ground for 15 min. After that, 100 μL of deionized water was added, and the mixture was ultrasonicated for 10 min. Finally, the suspension was transferred to a 1.5-mL microtube and centrifuged at 12,000 rpm for 5 min. The supernatant was collected for testing. The spiked solutions with arginine concentrations of 0.1, 1.0, 2.0, 5.0 and 10 mM were prepared by dissolving the compound in the supernatant. The cell sap without deionized water added was used for the determination of the average concentration of arginine. Single-cell Sampling. The holly leaves and Allium cepa bulbs were cleaned with deionized water. The holly leaves were sliced to obtain the cross-section of cells. A monolayer of inner epidermal cells was directly removed from the bulb and mounted smoothly onto a clean microscope glass slide. The pressure-assisted micro-sampling probe was controlled by a motorized micromanipulator (MP-225, Sutter Instrument, Novato, CA, USA) to accurately insert into the target cell. It was attached to a pico-liter pump (LPP01-100 Pico-liter Pump, Longer Precision Pump Co., Ltd) (For the instruction of pico-liter pump see Figure S-2). The pico-liter pump was used to suck the liquid samples under a negative pressure with a GlassTips probe. The gas pressure and the duration time were


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Analytical Chemistry both fixed to obtain the sampling volume. All the operations were observed with a microscope (Olympus BX51). When approximately 5 pL (holly leaves cells) or 20 pL (Allium cepa cells) of cell sap had been withdrawn from the cell, the probe was taken away from the cell and the harvested sap assayed by HFDI-MS. Mass Spectrometry Experiments. The quantitative analysis was carried out using a Thermo TSQ Quantum Access triple-quadrupole mass spectrometer (Thermo Fisher Scientific, Waltham, MA). The monitored conditions were optimezed by HFDI-MS in a positive mode as follows: for methyl salicylate, m/z 153→121, collision energy of 15 eV, tube lens of 86 V; for arginine, m/z 175→116, collision energy of 25 eV, tube lens of 86 V. The instrument settings were as follows: scan width of 1.0 u, scan time of 0.2 s, Q1 width of 0.7 u, CID gas of 1.5 mTorr. The capillary temperature was set at 300 °C. No voltage was applied at the probe tip, and the hydrogen flame was sufficient to achieve both thermal desorption and proton-transfer ionization for the analytes. The HFDI-IM-MS experiments and accurate mass measurements were performed with an ion-mobility quadrupole time-of-flight mass spectrometer (IM-Q-TOF MS, Agilent Technologies, Santa Clara, USA). The MS inlet temperature was set at 325 °C and the capillary entrance voltage was 800 V. The flow of drying gas was controlled to a minimum and the mass resolution was 40,000 FWHM. The frame rate was optimized at 5 frame s-1. Nitrogen as the IM drift gas was maintained at approximately 4 Torr and 25 °C, while the drift voltage was fixed at 1200 V. The ion mobility resolution was ~60 td/Δt (drift time, full width at half maximum). The commercial ionization source of ESI was removed ahead of our experiments.

RESULTS AND DISCUSSION Figure 1 shows the essential features of the experimental sucking cell sap in a single plant cell and the subsequent hydrogen flame desorption ionization procedure. As shown in Figure 1a, two kinds of holly leaves were used as the test samples. The leaves were cut into small pieces and mounted smoothly onto a clean microscope glass slide for subsequent single-cell analysis (Figure 1b). The probe was controlled by a three-dimensional manipulator and inserted into a single cell to sucked cell sap. Inspired by the cell pressure probe developed for controlling the sampling volume at the single-cell level,29 we used a pico-liter pump to collect the sample from a single cell, and the obtained sampling volume was calculated (Figure 1c). The hydrogen flame provided adequate heat for the thermal desorption of analytes. The vapor-phase analytes then reacted with the reactive charged species (mainly protonated water cluster ions) to be ionized.41-43 Finally, the analyte ions were transferred into the MS inlet, and a clear extracted ion current (EIC) was obtained. Figure 1e shows the EIC for multiple replicates of 2 mM arginine standard solution in SRM mode. Mechanism of HFDI-MS. Flames of various sources can serve as an ionization source, such as a butane flame, oxyacetylene flame, and wooden matchstick flame.44-47 The combustion of fuel in flame plasma is an exothermic process which generates light, heat, and charged species.45 These highly reactive charged species, such as CHO+, CH2O+, NO+, NO2H+, (H2O)nH+, Na+, and K+, are potentially useful for analyte ionization through ion-molecule reactions (IMRs). However, the inevitable carbon residues or carbon

nanoparticles generated by flame induce contaminations to the instruments, further cause the serious background signals to the MS spectra. Compared with other flames, such as AFI44 and FAPCI45, the hydrogen flame mainly generates protonated water cluster ions and few reactive charged species, introducing fewer contaminants into the mass spectrometer, which makes this flame friendlier to the mass spectrometric analysis. In HFDI-MS, the analytical process included two-step desorption-ionization. First, the hydrogen flame provided adequate heat to effectively eject and vaporize the analytes from the tip without inducing significant chemical degradation. Second, the vapor-phase analytes entered into a flame plasma plume and reacted with the reactive charged species to form analyte ions at atmospheric pressure. This ionization process was similar to FAPCI, in which the analyte was not directly exposed to the flame but was introduced into the ionization region.45 The reactive charged species were further captured and identified as protonated water cluster ions by MS (see Figure S-3 and Table S-1). These protonated water cluster ions served as proton donors, and through the PTR, they protonated the analyte molecules that had a higher proton affinity (reaction 2). The results showed that the HFDI-MS had a higher ionization efficiency for the small molecules covering a wide range of polarity. Compared with electrospray-based ionization methods, this method was more suitable to ionize compounds with low polarity and volatility, such as cinene and 4-methylstyrene (see Figure S-4). The few reactive charged species (Na+, K+) could be useful for analyte ionization through IMRs. Since the flash thermal desorption and proton-transfer ionization processes were separated in both time and space, it was favorable to alleviate the matrix effect for the quantitation of analytes in complex samples. H3O+ + (n-1)H2O

[(H2O)nH]+

(1)

[(H2O)nH]+

MH+ + nH2O

(2)

+

M

The M referred to the analyte molecule. Parameter Optimization. A series of experiments were performed to develop a robust approach for pressure-assisted micro-sampling probes coupled with hydrogen flame desorption ionization to realize the quantitative analysis of pico-liter volumes of metabolites from a single cell. First, the temperature of the hydrogen flame was optimized to achieve the best thermal desorption of the analytes and avoid damaging the probe tip. The temperature of the hydrogen flame assisted by carbon dioxide (VH2/VCO2=1:1) could be regulated in a large range, especially in the lower temperature zone compared with other flames (Figure 2c). The color of the hydrogen flame assisted by carbon dioxide was light blue, which made it easy to observe and control, while the color of the hydrogen flame assisted by nitrogen or helium was colorless. Moreover, to evaluate metabolites thermal stability, lysine, arginine and methyl salicylate served as the test samples. On the basis of experimental results (see Fiugre S-5) and the desorption temperature of most organic compounds, an ideal thermal desorption temperature for the analytes or metabolites in our experiment was about 350 °C (the size of the flame was 4-5 mm) (Figure 2a).48,49 Thermal imaging of the hydrogen flame was obtained by an infrared thermal imager (FLIR SC620) (Figure 3a), and the temperature at the top of the hydrogen flame was acquired three times by contacting the thermocouple. To enhance the ion transmission efficiency and avoid the damage of the tip caused by



scratching the MS inlet, the optimal distance of the tip to MS inlet was found to be 1 mm (Figure 2b). These experiments were conducted using the highest-grade borosilicate glass probe with a diameter of 2.5±0.5 μm (Figure 3b-d). The sucked volume in the tip could be controlled from pico-liters to nano-liters (see Figure S-6). When the flame touched the tip of the probe, the analytes were rapidly desorbed and formed a spray plume (Figure 3e). At the same time, the weight of the probe was evaluated before and after thermal desorption of the sample (see Table S-2). The results showed that most of the sample volume (weight) was thermally desorbed. To avoid the inaccuracy caused by the small amounts of analytes possibly adhering to the outer wall of the probe when sucking the analytes, the probe was immersed into the sample solution or a single cell at the same depth for every test. The results showed that the signal intensity (peak area) of lysine (m/z 147) increased linearly (R2 = 0.999) with the sucked volume (10-500 pL) under the optimal conditions of HFDI-MS (Figure 2d), which provided the possibility and prerequisite for the MS quantitation.

Figure 3. (a) Thermal imaging of the hydrogen flame (VH2/VCO2=1:1). (b) and (c) SEM images of the tip of the probe. (d) Microscope image of the probe post-sampling. (e) Photograph of the thermal desorption spray plume.

Quantitative Analysis. Varying concentrations of methyl salicylate standard solution were analyzed to obtain the limit of detection (LOD) and limit of quantitation (LOQ) for the present methodology by HFDI-MS (see Table S-3). The LOD and LOQ for methyl salicylate were determined as the signal-to-noise ratios (S/N) above 3 and 10 in the SRM mode, respectively. The results showed that the LOD was 8 μM (100 pL) (see Figure S-7), and that the LOQ was 40 μM (400 pL). Then, the experiments were conducted by analysis of methyl salicylate in a range of 0.1-10 mM. Analytes (400 pL) from the tip of the probe were rapidly ejected and ionized by a hydrogen flame. The signal of methyl salicylate (m/z 153 → 121) was detected in SRM mode (MS/MS of m/z 153; see Figure S-8). Figure 4a shows an EIC for six consecutive analyses of the methyl salicylate solution with a concentration of 2 mM using the optimal operating parameters for HFDI-MS. The peak areas from the EIC were used to evaluate the reproducibility. The relative standard deviation (RSD) calculated from the six replicates was less than 11% (n = 6 per concentration level). The abundance of the methyl salicylate was obtained by integrating the peak area under the corresponding EIC and used to generate the calibration curve (blue line) shown in Figure 4b. A linear curve (y= 484.3x + 284.9) was obtained in the concentration range of 0.1 to 10 mM with the correlation coefficient of R2 = 0.999. Then, the effect of different matrices on this method was further

investigated. The cell sap of the holly leaf served as the matrix solution for studying the signal intensity of methyl salicylate. The calibration curve was plotted by calculating the peak area of EIC for each concentration (0.1-10 mM). Figure 4b shows the calibration curve (red line) (y= 458.4x + 308.9) of methyl salicylate in the extracted cell sap matrix in the range of 0.1 to 10 mM with a correlation coefficient of R2 = 0.999. The results showed that the slope of the two calibration curves between the ethanol solution and matrix solution was not significantly different. The slope ratio (k2/k1) value was approximately 0.95, indicating that the matrix effect could be neglected for the quantitative analysis of methyl salicylate in the cell sap matrix.

Figure 4. (a) EIC for six consecutive methyl salicylate (2 mM) analyses via pressure-assisted micro-sampling probe HFDI-MS and (b) two linear curves for methyl salicylate in ethanol solution (R2 = 0.999) and matrix solution (R2 = 0.999) with a concentration range of 0.1−10 mM, respectively. The error bars represent the standard deviation of samples analyzed in sextuplicate.

Methyl salicylate, a common agent capable of eliciting chemical defense, was derived from the branches and leaves of holly plants. The compound was normally detected by headspace gas chromatography mass spectrometry (see Figure S-9). After that, the pressure-assisted micro-sampling probe coupled with HFDI-MS was used for direct quantitation of methyl salicylate in a single cell of holly leaves without introducing an internal standard. To improve the sensitivity, SRM scan mode was used to detect the methyl salicylate signal (m/z 153→121). The 400 pL of 1 mM methyl salicylate solution was sucked into the probe. The HFDI-MS analysis of the analytes produced EIC spectra in the SRM mode. The relative error of the average peak areas between the measured and theoretical value was 2.9%. Then, the EIC spectra of methyl salicylate were obtained for sucking 5 pL of the cell sap from an old-growth leaf cell and a tender leaf cell (see Figure S-10). We calculated the average (n=6) concentration of methyl salicylate in an old-growth leaf cell and a tender leaf cell to be 3.1 mM (RSD, 12%) and 0.72 mM (RSD, 19%), respectively. The results showed that there might be some differences and variations in the abundance of methyl


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Figure 5. HFDI-MS direct analysis of metabolites (a) in a single Allium cepa cell and (b) in the oil-filled pit of orange peels in the positive ion mode. Fructans molecular weights follow the rule: M=18+162n. DP is referred to degree of polymerization.

salicylate between different tissues and cells, which provided an important theoretical basis for extracting the methyl salicylate from plants. We also characterized and identified the metabolites in a single cell from a holly leaf (see Table S-4). In addition, this method was used for the direct quantitation of arginine in a single Allium cepa cell. Arginine was detected in the SRM mode (m/z 175→116; see Figure S-11). First, the LOD (10 μM, 100 pL) (see Figure S-12) and LOQ (50 μM, 400 pL) of arginine for this method were evaluated. Then, the two calibration curves for the standard solution (y= 552.5x + 126.8) and matrix solution (y= 506.3x + 138.7) were obtained in the concentration range of 0.1 to 10 mM (see Figure S-13 and Table S-5). The results (k2/k1, 0.92) showed that the low matrix effect had no significant effect on the direct quantitation of arginine. Next, the probe was used to suck approximately 20 pL of the cell sap for quantitative analysis of arginine in the SRM mode. The concentration of arginine in the inner epidermal cell was 0.11 mM (RSD, 11%; n=6) and the average concentration of arginine in the cell sap was approximately 0.20 mM (RSD, 3.4%; n=6). Other metabolites observed in the spectrum (Figure 5a) were identified based on the exact mass and the related literature (see Table S-6).25,50 Moreover, the technique of capillary micro-sampling combined with ESI-IMS-MS has been established to enhance the ion coverage and distinguish structural isomers at the single-cell level.22 It enlightened us to explore the application of HFDI-MS in this field. Therefore, we combined this method with the IMS to characterize and distinguish the relative abundance of two polymethoxyflavone isomers (SIN and TAN) in orange fruits. The structural identification of each isomer was confirmed through calculation of the collision cross-section values (see Table S-7). Then, 500 pL of the cell sap was sucked from the oil-filled pit and was analyzed by HFDI-IM-MS (Figure 5b and Table S-8). The results showed

that the concentrations of TAN and SIN in all three parts of the orange fruit all were significantly different (see Figure S-14), indicating that the distribution of TAN and SIN might be related to the variety, different regions, and fruit maturity.51,52 Finally, 500 pL of the cell sap was sucked for quantitative analysis of TAN and SIN. As a result, the average concentrations of TAN and SIN in the yellow orange peel were 22.3 μg/mL (RSD, 2.8%; n=3) and 3.5 μg/mL (RSD, 1.6%; n=3), respectively. The linearities, LODs, and LOQs of TAN and SIN were shown in Table S-9 and Figure S-15.

CONCLUSION In summary, a pressure-assisted micro-sampling probe coupled with HFDI-MS was developed for the direct quantitation of analytes at the pico-liter level or in a single cell without adding an internal standard. This workflow showed the advantages of 1) accurate sampling by a pico-liter pump, 2) quantitative thermal desorption for a small sample volume in the probe tip, 3) effective proton-transfer ionization, and 4) fewer matrix effects for small molecules, especially for volatile compounds. Compared with conventional electrospray-based analytical methods, this workflow could decrease ion suppression and provided a new perspective for the direct quantitation of metabolites at the single-cell level.

ASSOCIATED CONTENT Supporting Information The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.anal-chem.

AUTHOR INFORMATION Corresponding Author



*E-mail: [email protected]. Tel.: +86-021-54925300 (Guo, Y. L.)

Author Contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT We thank the National Natural Science Foundation of China for financial support (Grant Nos. 21532005, 21472228 and 21874144) and the National Key Research and Development Program of China (No. 2016YFC0800704).

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