Single Cell Analysis with Probe ESI-Mass Spectrometry: Detection of

Mar 18, 2014 - Multiple reaction monitoring (MRM)-profiling for biomarker discovery applied to human polycystic ovarian syndrome. Fernanda B. Cordeiro...
0 downloads 0 Views 3MB Size
Download PDF
Article pubs.acs.org/ac

Single Cell Analysis with Probe ESI-Mass Spectrometry: Detection of Metabolites at Cellular and Subcellular Levels Xiaoyun Gong, Yaoyao Zhao, Shaoqing Cai, Shujie Fu, Chengdui Yang, Sichun Zhang, and Xinrong Zhang* Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Department of Chemistry, Tsinghua University, Beijing China S Supporting Information *

ABSTRACT: Molecular analysis at cellular and subcellular levels, whether on selected molecules or at the metabolomics scale, is still a challenge now. Here we propose a method based on probe ESI mass spectrometry (PESI-MS) for single cell analysis. Detection of metabolites at cellular and subcellular levels was successfully achieved. In our work, tungsten probes with a tip diameter of about 1 μm were directly inserted into live cells to enrich metabolites. Then the enriched metabolites were directly desorbed/ionized from the tip of the probe for mass spectrometry (MS) detection. The direct desorption/ ionization of the enriched metabolites from the tip of the probe greatly improved the sensitivity by a factor of about 30 fold compared to those methods that eluted the enriched analytes into a liquid phase for subsequent MS detection. We applied the PESI-MS to the detection of metabolites in single Allium cepa cells. Different kinds of metabolites, including 6 fructans, 4 lipids, and 8 flavone derivatives in single cells, have been successfully detected. Significant metabolite diversity was observed among different cells types of A. cepa bulb and different subcellular compartments of the same cell. We found that the inner epidermal cells had about 20 fold more fructans than the outer epidermal cells, while the outer epidermal cells had more lipids. We expected that PESI-MS might be a candidate in the future studies of single cell “omics”.

H

spectrometry (LAESI-MS) has also been developed to accomplish single cell analysis.22 Further improvements were made in recent years to enable LAESI-MS for the subcellular detection of metabolites in single Allium cepa cells.9 Metabolite gradients within single A. cepa cells were successfully observed after piercing and cutting the cell wall away using a microdissection needle and then detecting by LAESI. Directly sucking a small amount of cell content into a nanospray tip for subsequent MS detection offers another way for live cell analysis.23,24 Till now, however, few methods had made use of an enrichment and separation procedure in single cell analysis, which might greatly improve the sensitivity. Probe ESI (PESI) offered such an opportunity. ESI has been generated on different solid bases, including papers,25,26 wooden tips,27 and surface-modified glass rods28 et al. Specially, Hiaroka proposed the concept of “probe electrospray ionization (PESI)”29 and thoroughly investigated the physical properties of this technique.30−32 In addition, Hiaroka applied PESI in the analysis of several biological systems, such as plant and animal tissues.33,34 However, no work about single cell analysis using probe ESI has been reported. Compared with conventional MS-based single cell methods, probe ESI has its distinctive potentials in single cell analysis: (1) direct sampling in live cells,

eterogeneity of biological systems causes distribution gradients of metabolites among different cells and even different subcellular compartments.1−3 Adjacent cells or subcellular compartments may have notable differences in composition.4,5 To better understand the complicated biological phenomena, methods are needed for the detection of metabolites at the cellular level6−8 or even down to the subcellular level.9,10 For decades, fluorescence microscopy has played a prominent role in single cell analysis.6,8,11 It has the outstanding capability of living cell imaging and offers high contrast targeted molecule specific images. Lately, great breakthrough has been made in super-resolution imaging with subcellular resolution by fluorescence microscopy.11−13 Regardless of its distinctive advantages, the detection capability of fluorescence microscopy relies on its molecular probes or reporters, which may have low specificity and have to be preselected. Furthermore, the labeling of a giving molecule may influence its location and biological function.6,8 Recently, mass spectrometry (MS)-based single cell analysis methods have been developed, which do not require labeling of targeted molecules and are suitable for the detection of unknown molecules. Matrix-assisted laser desorption ionization (MALDI) 14−17 and secondary ion mass spectrometry (SIMS)18−21 are the emerging methods in single cell analysis. These two methods are generally operated under vacuum conditions and are not suitable for live cell analysis. Besides MALDI and SIMS, laser ablation electrospray ionization mass © 2014 American Chemical Society

Received: November 21, 2013 Accepted: March 18, 2014 Published: March 18, 2014 3809

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

Article

Figure 1. Schematic of the probe ESI setup. (a) Microscopic image of the sampling procedure. Monolayer A. cepa cells were placed smoothly on a glass slice. The dark cells in the image were alive, with certain amounts of pigment in their vacuoles, while the light ones were dead. Only live cells were used in our experiment. (b) Setup of the coupling with MS. A DC high voltage (+2.5 kV for the positive mode and −2.6 kV for the negative mode) was applied to the probe. Assistant solvents were directly sprayed onto the tip of the probe to generate electrospray.

probes were washed with methanol/water (1:1), ultrapure water, and ethanol, respectively. Afterward, the probes could be used again. Sampling and Detection. The probe was controlled by a three-dimensional manipulator (MP-225, Sutter Instrument Company) to precisely insert into targeted cells. The smallest microstep size of the manipulator was 65 nm. All the operations were observed with a microscope (Olympus IX81). After insertion, the probe was kept in the cell for 30 s to enrich metabolites. Then, the probe was taken away from the cell and accurately positioned in front of the MS inlet with a distance of 5 mm for desorption/ionization (Figure 1b). The tip of the probe was then wetted by sprayed-assistant solvents, which was accompanied by undergoing desorption/ionization at an applied high voltage (+2.5 kV for positive mode and −2.6 kV for negative mode) for MS detection. Analytes that had been enriched on the tip were then desorbed/ionized from the tip and transferred into MS. A similar desorption/ionization method could be found in literature.33 In the optimization experiments, all the measurements were repeated over three times to be certain of the results. In the single-cell experiments, the measurements were repeated in over five different individual cells of the same type to be certain of the results. Mass Spectrometry. Accurate mass measurements were accomplished on Orbitrap MS (Q-Exactive, Thermo Scientific, San Jose, CA). Capillary temperature: 320 °C, tube lens voltage: 50 V, mass resolution: 70000, maximun inject time: 50 ms, and microscans: 1. The commercial ionization source of ESI was removed ahead of our experiments. Other MS experiments were accomplished on LTQ MS (Thermo Scientific, San Jose, CA). Capillary temperature: 275 °C, capillary voltage: 9 V, tube lens voltage: 100 V, maximum inject time: 200 ms, and microscans: 2. The commercial ionization source of ESI was removed ahead of our experiments. Metabolites Identification. Metabolites identification was based on the accurate mass measurements and isotopic profiles. Besides these two ways, MS/MS experiments were also carried out to help identify the potential structures of metabolites (MS/MS results of some typical metabolites in A. Cepa cells were shown in Figures S6−S10 of the Supporting Information). Furthermore, databases (Plant Metabolic Network database: http://plantcyc.org/; or LIPID MAPS: http://www.lipidmaps. org/) and literature22,35,36 were also taken into account for verification.

(2) enrichment of metabolites, which improved the sensitivity, (3) subcellular detection could be achieved with miniaturized probes, (4) reduced disturbance to the original distributions of metabolites during sampling, and (5) simplified device and reusable probes. It would be of great significance to introduce probe ESI into the field of single-cell analysis. Herein, we demonstrate single-cell analysis with PESI-MS for the detection of metabolites at the cellular and subcellular levels. Tungsten probe with a tip diameter of 1 μm was directly inserted into live cells to enrich metabolites. A hyphenated interface between the probe and MS was designed to allow for direct desorption/ionization of the enriched analytes from the tip of the probe and detected by MS. The enrichment of metabolites on the probe and direct desorption/ionization of them into MS greatly improved the sensitivity. We applied this method to the detection of metabolites in single A. cepa cells. Multispecies of metabolites in single A. cepa cells were successfully detected. Significant metabolite diversity was observed among different cells types of the A. cepa bulb and different subcellular compartments of the same cell.

■

EXPERIMENTAL SECTION Chemicals and Sample Preparation. Phosphatidylcholine PC (34:1), maltohexaose, maltoheptaose, and HPLC-grade methanol were purchased from Sigma. Angiotensin II (≥99.0%) and somatostatin (≥99.0%) were purchased from Shanghai Chutai Biological Technology Company, Ltd. (China) and chloroform (A.R.) and N,N-dimethylformamide (DMF, A.R.) were purchased from Sinopharm Chemical Reagent Company, Ltd. Purple A. cepa bulbs were purchased from a local store in Beijing, China and stored at 4 °C before use. The A. cepa bulbs were washed with ultrapure water (Barnstead Nanopure, Thermo Scientific, San Jose, CA., resistance ≥ 18 MΩ cm−1) to clean the surface of the epidermis. A monolayer of epidermal cells were directly removed from the bulb and mounted smoothly onto a clean microscope glass slide for subsequent experiments. Probe Preparation. The probe was designed to be with a tip diameter of ≤1 μm and controlled by a three-dimensional manipulator to precisely insert it into the targeted cell (Figure 1a). The tungsten-made probes (99.9% tungsten, ST-20-5, purchased from GGB Industries Inc.) were first washed with methanol/water (1:1) to clean its surface and then soaked in 5% HNO3 for 5 min. Afterward, the probes were washed with ultrapure water, methanol/water (1:1), and ethanol, respectively. Regeneration of the probes was quite simple. The used 3810

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

Article

Figure 2. Investigation of the detection capability of our method compared with that of conventional nano-ESI. (a) Mass spectrum of 1 μM angiotensin II aqueous solution obtained using our method. The enrichment time was 15 s. The assistant solvent for sample desorption/ionization was methanol/water = 1:1. Angiotensin II was represented by its singly protonated ion m/z 1046 and single sodium adduct ion m/z 1068 in the spectrum. (b) Nano-ESI mass spectrum of the eluted angiotensin II solution from the probe after enrichment. The elution solvent was methanol/ water = 1:1. The enrichment procedure was the same as in (a).

Analytes Enrichment and Selective Detection of the Enriched Analytes. The amount of captured analytes increased with enrichment time. To investigate the influence of enrichment time, an aqueous solution of angiotensin II with a concentration of 0.5 μM was used as the sample. The probe was immersed into the solution for different periods of time to enrich angiotensin II. As was shown in Figure 3, the obtained signal intensity of angiotensin II increased along with extended enrichment time from 5 to 300 s, and the signal-to-noise ratio was greatly improved. The signal intensity obtained at 300 s was over 20 fold stronger compared to that obtained at 5 s. This confirmed the enrichment feature of the probe and also indicated that the sensitivity of probe ESI could be greatly improved with extended enrichment time. Selective detection of the captured analytes on the tip of the probe could be achieved with appropriate assistant solvents, eliminating the interferences from unwanted species. To further look into this issue, an aqueous mixture solution containing five different species was prepared as the sample (Detailed content of the solution was described in Table S1 of the Supporting Information). Three different assistant solvents with different polarity were prepared for the detection (details were shown in Figure 4). The obtained mass spectra were shown in Figure S1 of the Supporting Information. In accordance with the results, the two hydrophilic sugars were better detected under hydrous solvents, with the best signals given by methanol/water. On the contrary, the two hydrophobic species of somatostatin and phosphatidylcholine PC(34:1) could hardly be detected under methanol/water. They were easily detected by methanol/ water/chloroform and DMF/acetonitrile. The anhydrous solvent DMF/acetonitrile gave the best signals of somatostatin and PC(34:1). The key point seemed to be the diverse solubility of analytes in assistant solvent. Analytes that had better solubility in the used assistant solvent would be more easily desorbed/ionized, while those analytes that were hardly soluble in the used assistant solvent tended to remain on the probe. Angiotensin II was further used as an internal standard. MS signal intensities of the other four species were compared with that of m/z 1046 to get relative intensities, as was illustrated in Figure 4. It was obvious that the hydrophilic sugars showed decreased signal intensity with the decreased solvent polarity. The signal intensity of the sugars under methanol/water was about 7 fold higher than that under DMF/acetonitrile. In comparison, hydrophobic somatostatin and PC(34:1) showed

Cell Staining. Cell staining was only used in the experiment that aimed at confirming the ability of our method in subcellular detection. Methylene blue was dissolved in methanol/water = 1:1 in a concentration of 0.1%. Epidermal cells were immersed in the dye solution for one minute. After staining, the cells were washed by ultrapure water and then mounted on a glass slide for detection. Safety Considerations. When experiments were performed, high DC voltage was applied to the tip of the syringe. Actions for insulation of both the operator and the MS with the high voltage source should be taken to avoid danger.

■

RESULTS AND DISCUSSION

Considering the fact that mammalian cells generally have a size of ∼10 μm (plant cells are even larger), the tip of the probe was designed to be ∼1 μm so that subcellular detection of metabolites was possible. The schematic setup was illustrated in Figure 1. The probe was controlled by a three-dimensional manipulator to precisely insert into the targeted cell for the enrichment of metabolites. Then, the probe was taken away from the cell and placed in front of the MS inlet. After wetting the tip with solvents, a high voltage of about 2500 V was applied to the probe. The analytes enriched on the tip were then desorbed/ionized directly and transferred into MS for detection. (Detailed information was shown in Experimental Section.) The sensitivity of direct desorption/ionization from the probe was compared with conventional nano-ESI after elution of the captured analytes from the probe into the liquid phase.37 An aqueous solution of 1 μM angiotensin II was used as the sample. In accordance with the result in Figure 2a, our method successfully detected angiotensin II with a strong signal intensity. The singly protonated ion (m/z 1046) and single sodium cation (m/z 1068) adduct of angiotensin II could clearly be observed in the spectrum of Figure 2a. In comparison, if the captured angiotensin II on the probe was eluted into a 1 nL solution and subsequently detected by nanoESI, the signal intensity was poor, due to the dilution caused by elution (Figure 2b). Only the singly protonated ion could be recognized. Our method showed significant enhanced signal intensity by a factor of about 30 fold. Direct desorption/ ionization of the enriched analytes from the probe successfully overcame the shortage of dilution in conventional nano-ESI methods and greatly improved the sensitivity, making probe ESI more suitable for single cell analysis. 3811

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

Article

Figure 3. (a−h) Mass spectra obtained under different enrichment time. The sample was 0.5 μM of angiotensin II aqueous solution. With longer enrichment time, the signal intensity was obviously enhanced. Angiotensin II was represented by its singlely protonated ion m/z 1046, single sodium adduct ion m/z 1068, and double sodium adduct ion m/z 1090 in the spectra. (i) Comparison of the MS signal intensity of angiotensin II obtained with different enrichment time.

Tolerance to High Viscosity and Salinity. The tolerance of PESI-MS to high viscosity and salinity was also examined, considering the fact that cytoplasm was actually viscous and

increased signal intensity with the decreased solvent polarity. Their signal intensity under DMF/acetonitrile was about 15 fold higher than that under methanol/water. 3812

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

Article

Figure 4. Relative signal intensities of somatostatin, maltohexaose, maltoheptaose, and PC(34:1) compared to angiotensin II under different assistant solvents. (a) Relative signal intensity of the two hydrophilic sugars, maltohexaose and maltoheptaose and (b) relative signal intensity of the two hydrophobic species, somatostatin and PC(34:1).

Figure 5. Metabolite diversity of two different kinds of A. cepa cells revealed by their mass spectra. (a) Actual picture of an A. cepa bulb layer, (b) microscopic image and the corresponding mass spectra of an inner epidermal cell, and (c) microscopic image and the corresponding mass spectra of an outer layer cell. The left row of mass spectra was in the negative mode, and the right row was in the positive mode. The enrichment time was 30 s, and the assistant solvent was methanol/water/chloroform = 3:1:1. For substance identification, see Table S2 of the Supporting Information.

II dissolved in physiological saline. The signal intensity of our method was about 6 fold higher than that of nano-ESI. Apparent matrix peaks could be found in the spectrum of nanoESI, while few matrix peaks could be found in the spectrum of our method. Quantitative Analysis. Quantitative analysis could be achieved in our method by adding an appropriate internal standard in the assistant solvent. To confirm this, we quantified the concentration of sucrose in A. Cepa bulb. With consideration that all fructans in the A. Cepa bulb are polymerized by C6 saccharides, arabinose (C5 saccharide) was used as the internal standard to avoid interferences from the fructans in the A. Cepa bulb. Standard solutions of sucrose with concentrations of 10−300 mM were used to establish the standard curve (Figure S4 of the Supporting Information). The signal intensity of sucrose was compared to arabinose to give a

salty sample. Intact egg yolk was used as the highly viscous sample. Detection results were shown in Figure S2 of the Supporting Information. Our method could easily accomplish the detection in both positive and negative modes. Many components of egg yolk were detected, including lipids, amino acids, and fatty acids, etc. In comparison, we tried to use conventional nano-ESI to detect the components of intact egg yolk, but no signal was obtained. It seemed that the highly viscous yolk blocked the tip of nano-ESI, and it could not be sprayed out even at very high voltage of 4000 V. To demonstrate the tolerance to highly salty sample, an aqueous solution with 20 μM angiotensin II and 0.154 M NaCl (physiological saline) was prepared as the sample. Detection results of our method and conventional nano-ESI were compared, as was shown in Figure S3 of the Supporting Information. Both methods were able to detect the angiotensin 3813

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

Article

Figure 6. Slight composition differences distinguished between two adjacent outer epidermal cells. The main components of the two outer epidermal cells were the same but with different relative abundance.

relative intensity (Isucrose/Iarabinose). Finally, the concentration of sucrose in the A. Cepa bulb was detected to be 20−50 mM. This result matched well with literature (10−80 g/g fresh weight).38 Single Cell Analysis. We applied our probe ESI in single A. cepa cell analysis. Two different kinds of cells, the outer epidermal cells and the inner epidermal cells, were analyzed at the cellular level (Figure 5). Significant differences of metabolite species between the two kinds of cells have been observed. For example, six fructans (DP4-DP9, degree of polymerization) were assigned in inner epidermal cells (m/z 705, 867, and 1029 in the positive spectrum and m/z 665/683, 827/845, 1007, 1169, 1331, 1493 in the negative spectrum; see Figure 5b), while only three fructans (DP4-DP6) were assigned in the outer epidermal cells (m/z 705, 867 and 1029; see Figure S5 of the Supporting Information), indicating that inner epidermal cells had more fructan species than outer epidermal cells. Although both inner and outer epidermal cells had fructan DP4-DP6, these three fructans seemed to be the major species in inner epidermal cells, while in outer epidermal cells they seemed to be less abundant (Figure 5f and Figure S5 of the Supporting Information). Different lipid profiles were also observed between the two kinds of cells. Three kinds of lipids were detected in outer epidermal cells, including two PCs (m/z 796 and 820), two phosphatidyl ethanolamines (PEs, m/z 758, 782), and one flavone derivative (m/z 665) in the positive spectrum with high abundance. In comparison, only two flavone derivatives (m/z 661 and 759, in negative mode) were observed in inner epidermal cells. The results above showed that the outer epidermal cells were rich in lipids, while the inner epidermal cells were rich in fructans. We also compared the chemical compositions of adjacent outer epidermal cells (Figure 6). The most abundant species of the two outer epidermal cells were the same, including two PCs, two PEs, and one flavone derivative but with a slight difference in relative abundance. In cell A, the two PCs had a higher abundance (m/z 796 and 820) than the flavone derivative (m/z 665), while in cell B, the abundances were reversed. This result revealed the heterogeneity between adjacent cells. Subcellular Analysis. To demonstrate the subcellular detection capability of our method, a nucleus dye, namely methylene blue, was used to stain the outer epidermal cells (Figure 7a). The nuclei of the cells were deeply stained by methylene blue, as could be seen from the microscopic image in

Figure 7. Cell staining experiment. (a) Microscopic image of a stained outer epidermal cell using methylene blue, (b) mass spectrum of the cytoplasm, and (c) mass spectrum of the stained nucleus. The enrichment time was 10 s, and the assistant solvent was methanol/ water = 1:1. Methylene blue was represented by m/z 284 (positive mode) in the mass spectrum.

Figure 7a. We compared the detection results of the nucleus and the cytoplasm of the same cell (Figure 7, panels b and c). Significantly higher abundance of methylene blue was found in the nucleus than that in the cytoplasm, according to the mass spectra. The signal intensity of methylene blue in nucleus was about 20 fold higher than that in the cytoplasm. The results given by our SC-SPME-MS method matched well with the microscopic image. This experiment confirmed the subcellular detection capability of our probe ESI. Finally, subcellular analysis was carried out on outer epidermal cells (Figure 8). In accordance with the spectra, four kinds of flavone derivatives were observed in the nucleus (m/z 463, 499, 625, and 661; see Figure 8c). The cytoplasm had a more complex chemical composition. The four flavone derivatives that were already observed in the nucleus were also found in cytoplasm but with different abundances (Figure 8b). Apart from the four species, other peaks of m/z 927, 1089, and 1251 were also observed, which had already been found in cellular analysis. The results clearly indicated that molecule distribution within a single cell was heterogeneous. 3814

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

■

Article

ACKNOWLEDGMENTS This research is supported by the 973 program (Grant 2013CB933800), the National Natural Science Foundation of China (Grants 21390411 and 21125525), and the Ministry of Science and Technology of China (Grants 2011YQ090005 and 2011YQ6008402). We sincerely thank our teammates Song Wang and Yanyan Li for their help in the experiments.

■

(1) Ovadi, J.; Saks, V. Mol. Cell. Biochem. 2004, 256−257, 5−12. (2) Nat. Chem. Biol. 2010, 6, 691. (3) Burton, R. A.; Gidley, M. J.; Fincher, G. B. Nat. Chem. Biol. 2010, 6, 724−732. (4) Rubakhin, S. S.; Romanova, E. V.; Nemes, P.; Sweedler, J. V. Nat. Methods 2011, 8, S20−29. (5) Lidstrom, M. E.; Konopka, M. C. Nat. Chem. Biol. 2010, 6, 705− 712. (6) Trouillon, R.; Passarelli, M. K.; Wang, J.; Kurczy, M. E.; Ewing, A. G. Anal. Chem. 2013, 85, 522−542. (7) Avery, S. V. Nat. Rev. Microbiol. 2006, 4, 577−587. (8) Lin, Y.; Trouillon, R.; Safina, G.; Ewing, A. G. Anal. Chem. 2011, 83, 4369−4392. (9) Stolee, J. A.; Shrestha, B.; Mengistu, G.; Vertes, A. Angew. Chem., Int. Ed. 2012, 51, 10386−10389. (10) Glory, E.; Murphy, R. F. Dev. Cell 2007, 12, 7−16. (11) Stender, A. S.; Marchuk, K.; Liu, C.; Sander, S.; Meyer, M. W.; Smith, E. A.; Neupane, B.; Wang, G.; Li, J.; Cheng, J. X.; Huang, B.; Fang, N. Chem. Rev. 2013, 113, 2469−2527. (12) Kner, P.; Chhun, B. B.; Griffis, E. R.; Winoto, L.; Gustafsson, M. G. Nat. Methods 2009, 6, 339−342. (13) Huang, B.; Babcock, H.; Zhuang, X. Cell 2010, 143, 1047−1058. (14) Boggio, K. J.; Obasuyi, E.; Sugino, K.; Nelson, S. B.; Agar, N. Y.; Agar, J. N. Expert Rev. Proteomics 2011, 8, 591−604. (15) Yang, J.; Caprioli, R. M. Anal. Chem. 2011, 83, 5728−5734. (16) Rubakhin, S. S.; Sweedler, J. V. Anal. Chem. 2008, 80, 7128− 7136. (17) Amantonico, A.; Urban, P. L.; Fagerer, S. R.; Balabin, R. M.; Zenobi, R. Anal. Chem. 2010, 82, 7394−7400. (18) Lanni, E. J.; Rubakhin, S. S.; Sweedler, J. V. J. Proteomics 2012, 75, 5036−5051. (19) Monroe, E. B.; Jurchen, J. C.; Lee, J.; Rubakhin, S. S.; Sweedler, J. V. J. Am. Chem. Soc. 2005, 127, 12152−12153. (20) Szakal, C.; Narayan, K.; Fu, J.; Lefman, J.; Subramaniam, S. Anal. Chem. 2011, 83, 1207−1213. (21) Fletcher, J. S.; Rabbani, S.; Henderson, A.; Blenkinsopp, P.; Thompson, S. P.; Lockyer, N. P.; Vickerman, J. C. Anal. Chem. 2008, 80, 9058−9064. (22) Shrestha, B. V. A. Anal. Chem. 2009, 81, 8265−8271. (23) Mizuno, H.; Tsuyama, N.; Harada, T.; Masujima, T. J. Mass. Spectrom. 2008, 43, 1692−1700. (24) Lorenzo Tejedor, M.; Mizuno, H.; Tsuyama, N.; Harada, T.; Masujima, T. Anal. Chem. 2012, 84, 5221−5228. (25) Liu, J. W. H.; Manicke, N. E.; Lin, J.; Cooks, R. G.; Ouyang, Z. Anal. Chem. 2010, 82, 2463−2471. (26) Wang, H.; Liu, J.; Cooks, R. G.; Ouyang, Z. Angew. Chem., Int. Ed. 2010, 49, 877−880. (27) Hu, B.; So, P. K.; Chen, H.; Yao, Z. P. Anal. Chem. 2011, 83, 8201−8207. (28) Jeng, J.; Shiea, J. Rapid Commun. Mass Spectrom. 2003, 17, 1709−1713. (29) Hiraoka, K.; Nishidate, K.; Mori, K.; Asakawa, D.; Suzuki, S. Rapid Commun. Mass Spectrom. 2007, 21, 3139−3144. (30) Mandal, M. K.; Chen, L. C.; Hashimoto, Y.; Yu, Z.; Hiraoka, K. Anal. Methods 2010, 2, 1905. (31) Mandal, M. K.; Chen, L. C.; Hiraoka, K. J. Am. Soc. Mass Spectrom. 2011, 22, 1493−1500. (32) Yoshimura, K.; Chen, L. C.; Asakawa, D.; Hiraoka, K.; Takeda, S. J. Mass Spectrom. 2009, 44, 978−985.

Figure 8. Metabolite diversity of subcellular zones obtained from an outer epidermal cell. (a) Microscopic image of an outer epidermal cell, (b) mass spectrum of the cytoplasm that was away from the nucleus, and (c) mass spectrum of the nucleus. The enrichment time was 30 s, and the assistant solvent was methanol/water/chloroform = 3:1:1. For substance identification, see Table S2 of the Supporting Information.

■

CONCLUSIONS In summary, we have demonstrated a probe ESI-MS method for single cell analysis. It allowed for direct sampling in live cells. The enrichment feature of the probe and direct desorption/ionization of metabolites from the SPME probe greatly improved the sensitivity. Selective detection of the enriched metabolites on the probe reduced the interference of unwanted species and simplified the spectra. Compared with conventional laser based and glass nanotip-based single cell analysis methods, our probe ESI offered a distinct way of sampling and detection. Besides the present work, there are still more that could be done to further improve the method. For example, appropriate surface modification of the probe might enable targeted enrichment of metabolites, thus significantly eliminating the interference of other species. In addition, the probe could be further miniaturized so that the disturbance to the content in cells would be reduced. With specially miniaturized probes, continuous monitoring of a live single cell might be achieved. Finally, in situ derivation of the enriched metabolites on the probe might further improve the sensitivity. We expected that probe ESI might be a powerful candidate in the future studies of single cell “omics”.

■

ASSOCIATED CONTENT

* Supporting Information S

Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org.

■

REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Tel: +86-10-6278 2485. Present Address

Department of Chemistry, Beijing Key Laboratory for Microanalytical Methods and Instrumentation, Tsinghua University, Beijing 100084, P.R. China. Notes

The authors declare no competing financial interest. 3815

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816

Analytical Chemistry

Article

(33) Mandal, M. K.; Saha, S.; Yoshimura, K.; Shida, Y.; Takeda, S.; Nonami, H.; Hiraoka, K. Analyst 2013, 138, 1682−1688. (34) Yu, Z.; Chen, L. C.; Suzuki, H.; Ariyada, O.; Erra-Balsells, R.; Nonami, H.; Hiraoka, K. J. Am. Soc. Mass Spectrom. 2009, 20, 2304− 2311. (35) Downes, K.; Terry, L. A. Talanta 2010, 82, 118−124. (36) Slimestad, R. F. T.; Vagen, I. M. J. Agric. Food Chem. 2007, 55, 10067−10080. (37) Mandal, M. K.; Yoshimura, K.; Saha, S.; Ninomiya, S.; Rahman, M. O.; Yu, Z.; Chen, L. C.; Shida, Y.; Takeda, S.; Nonami, H.; Hiraoka, K. Analyst 2012, 137, 4658. (38) Davis, F.; Terry, L. A.; Chope, G. A.; Faul, C. F. J. J. Agric. Food Chem. 2007, 55, 4299−4306.

3816

dx.doi.org/10.1021/ac500882e | Anal. Chem. 2014, 86, 3809−3816