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A Selective Imidazoline-2-Thione-Bearing Two-Photon Fluorescent Probe for Hypochlorous Acid in Mitochondria Qingling Xu, Cheol Ho Heo, Jin A Kim, Hye Sue Lee, Ying Hu, Dayoung Kim, Kunemadihalli Mathada Kotraiah Swamy, Gyoungmi Kim, Sang-Jip Nam, Hwan Myung Kim, and Juyoung Yoon Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.6b01738 • Publication Date (Web): 23 May 2016 Downloaded from http://pubs.acs.org on May 24, 2016

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Analytical Chemistry

A Selective Imidazoline-2-Thione-Bearing Two-Photon Fluorescent Probe for Hypochlorous Acid in Mitochondria Qingling Xu,†a Cheol Ho Heo,†b Jin A Kim,†a Hye Sue Lee,b Ying Hu,a Dayoung Kim,a Kunemadihalli Mathada Kotraiah Swamy,a,c Gyoungmi Kim,a Sang-Jip Nam,a Hwan Myung Kim*b and Juyoung Yoon*a a

Department of Chemistry and Nano Science, Ewha Womans University, Seoul 120-750, Korea

b

Department of Energy Systems Research, Ajou University, Suwon, Gyeonggi-do 443-749, Korea

c

Department of Pharmaceutical Chemistry, V. L. College of Pharmacy, Raichur-584 103, Karnataka State, INDIA Fax: (+82) 2-3277-2384 (J. Y.). E-mail: [email protected] (J. Y.) and [email protected] (H. M. K.).

ABSTRACT: Hypochlorite (OCl−) plays a key role in the immune system and is involved in various diseases. Accordingly, direct detection of endogenous OCl− at the subcellular level is important for understanding inflammation and cellular apoptosis. In the current study, a two-photon fluorescent off/on probe (PNIS) bearing imidazoline-2-thione as an OCl− recognition unit and triphenylphosphine (TPP) as a mitochondrial-targeting group was synthesized and examined for its ability to image mitochondrial OCl− in situ. This probe, based on the specific reaction between imidazoline-2-thione and OCl−, displayed a selective fluorescent off-on response to OCl− with the various reactive oxygen species in physiological medium. PNIS was successfully applied to the imaging of endogenously-produced mitochondrial OCl− in live RAW 264.7 cells via two-photon microscopy.

Reactive oxygen species (ROS) are involved in many important biological processes such as anti-inflammatory regulation, pathogen response, and aging.1 Among the various ROS, hypochlorite (OCl-) plays a key role in innate immunity and is typically produced by the myeloperoxidase (MPO)-catalyzed reaction of chloride ions and hydrogen peroxide in order to kill pathogens.2 OCl- not only inhibits inflammation but can also control cellular apoptosis.3-4 Conversely, OCl- can rapidly react with many biomolecules to cause various disorders including obesity, cancer, and diabetes.5 Accordingly, it is important to image OCl- in situ.6-23 Fluorescent probes have proven to be effective tools for monitoring biologically-relevant species in vitro and in vivo.24-29 Two-photon microscopy (TPM) displays many advantages over one-photon probes, such as greater tissue penetration depth and low phototoxicity.30-37 TPM-based small molecule probes have proven to be more useful tools compared with one-photon-based fluorescent probes, especially for tissue imaging. Mitochondria are known as major energy sources and are also the main source of ROS production, accumulation of which can cause cell death and various diseases.38,39 Thus, over the past few years, the imaging of OCl- in mitochondria has drawn a lot of attention.16-23 Recently, we reported that imidazoline-2-thiones are unique and selective reacting moieties for OCl- among the various ROS.15 In the current study, we synthesized a nov-

el imidazoline-2-thione derivative, PNIS, as a selective two-photon probe to image OCl- in mitochondria. PNIS contains a triphenylphosphine (TPP) functional group for mitochondrial targeting, which displays a selective fluorescence enhancement with OCl-. It is noteworthy that PNIS is soluble in 100% aqueous solution without the aid of organic solvents. The specific reaction of PNIS with OCl- yielded the corresponding fluorescent imidazolium product 5. PNIS was successfully applied to the imaging of endogenously-produced mitochondrial OCl− in live cells via two-photon microscopy. Scheme 1. Synthesis of the fluorescent probe PNIS and its reaction with OCl-.

(a) 2-(2-(2-iodoethoxy)ethoxy)ethanol, (b) Sulfur, Sodium methoxide/ethanol, (c) PPh3/CBr4, (d) PPh3/CH3CN

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Experimental Section General Methods. Unless otherwise noted, materials were obtained from commercial suppliers and used without further purification. Thin-layer chromatography (TLC) was carried out using Merck 60 F254 plates with a thickness of 0.25 mm. Chemical shifts are given in ppm and coupling constants (J) in Hz. Fluorescence Studies. Biologically-relevant reactive oxygen species (ROS) were added to DW for stock solutions. To check the fluorescence spectra, an excitation wavelength of 325 nm and a slit width of 5 nm/5 nm were used. The different concentrations of OCl- are explained in the fluorescence emission changes. Unless otherwise noted, all experiments were carried out using 2.0 µM PNIS in PBS buffer (50 mM, pH 7.4) at room temperature, with different levels of ROS. Generation of ROS/RNS. H2O2 was diluted from a 28% solution in water, and tert-butyl hydroperoxide was diluted from a 70% solution in water. ROO• was generated from 2,2'-azobis(2-amidinopropane)dihydrochloride, NO• was from SNP (sodium nitroferricyanide (III) dihydrate), and •OH was from the reaction of iron(II) chloride (200 μM) with H2O2 (400 μM). The above ROS or RNS were incubated with probe in KH2PO4 buffer (50 mM, pH 7.4) for 30 min. ONOO- was prepared according to previous literature, and the concentration was determined by the absorbance at 302 nm.40 NaClO was obtained by dilution of 5% solution in water. Cell Culture. Both cell lines were passaged and plated on glass-bottomed dishes (NEST) for two days prior to imaging. They were maintained in a humidified atmosphere of 5/95 (v/v) CO2/air at 37 °C. The cells were treated and incubated with 10 μM PNIS at 37 °C under 5% CO2 for 30 min, washed three times with phosphate buffered saline (PBS, Gibco), and subsequently imaged following further incubation in colorless serum-free medium for 30 min. The specific culture conditions were: MEM (WelGene Inc, Seoul, Korea) for HeLa human cervical carcinoma cells (ATCC, Manassas, VA, USA) and DMEM (WelGene Inc, Seoul, Korea) for RAW 264.7 cells (ATCC, Manassas, VA, USA), both supplemented with 10% FBS (WelGene), penicillin (100 units/ml), and streptomycin (100 μg/ml). Two-Photon Fluorescence Microscopy. Twophoton fluorescence microscopy images of PNIS-labeled cells were obtained with spectral confocal and multiphoton microscopes (Leica TCS SP8 MP), using a 40x oil objective and a numerical aperture (NA) of 1.30. The twophoton fluorescence microscopy images were obtained with a DMI6000B microscope (Leica) by exciting the probes with a mode-locked titanium-sapphire laser source (Mai Tai HP; Spectra Physics, 80 MHz pulse frequency, 100 fs pulse width) set at a wavelength of 700 nm and an output power of 1290 mW, which corresponded to an approximate average power of 1.41×109 mW/cm2 in the focal plane. Live cell imaging was performed using the live cell incubator system (Chamlide IC; Live Cell Instrument) to create a stable cell environment by long-term

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maintenance of the proper temperature, humidity, and pH. To obtain images in the 400-600 nm range, internal PMTs were used to collect the signals in 8-bit unsigned 512×512 and 1024×1024 pixel scans, respectively, at a speed of 400 Hz. Colocalization Experiment. Colocalization experiments were conducted in HeLa cells by co-staining with appropriate combinations of PNIS (10 μM) and MitoTracker Red FM (MTR, 1.0 μM) for 30 min. TPM and OPM images were obtained by collecting the emissions at 400550 nm (PNIS, λex = 700 nm) and 650-700 nm (MTR, λex = 552 nm), respectively. The background images were corrected, and the distribution of pixels in the TPM and OPM images, acquired in the green and red channels, were subsequently compared using a scattergram. The Pearson’s colocalization coefficient (A) was calculated using the LAS AF software. Synthesis of 2. 1 (0.102 g, 0.557 mmol) and 2-(2-(2iodoethoxy)ethoxy)ethanol (0.151 g, 0.580 mmol) in CH3CN o (10 mL) was refluxed in 95 C sand bath for 43 h. After cooling, solvent was removed and the residue dissolve in methanol (1 mL). Hexane was added to the solution and precipitation was collected, it was repeated 3 times to get product as 1 white solid (0.144 mg, 0.326 mmol, 58.5% ) H NMR (300 MHz, CDCl3): δ 10.54 (s, 1H), 8.25 (s, 1H), 8.08-8.02 (m, 3H), 7.60 (m, 2H), 4.86 (t, 2H), 4.28 (s, 3H), 4.14 (t, 2H), 3.82 (m, 4H), 3.67 (m, 4H), 3.53 (t, 1H). 13C NMR (75 MHz, CDCl3): δ 147.07, 131.61, 131.56, 130.80, 130.20, 128.43, 128.33, 127.15, 127.12, 110.80, 110.27, 72.70, 70.34, 70.02, 67.29, 61.18, 47.41, 34.27. LC/MS (ESI) m/z = 315.1727 [M]+, calc. for C18H23N2O3 = 315.1709. Synthesis of 3. Sodium methoxide (0.3 mL, 0.5 M, 0.15 mmol) was added to the flask of 2 ( 60 mg, 0.136 mmol) and sulfur (4.6 mg, 0.143 mmol) with methanol (unhydrous, 6 mL) heated by sand bath (70 oC). After stirring for 15 h, the mixture was cooled down and solvent was removed. CH2Cl2 was added, washed with H2O and dried over unhydrous MgSO4. Then CH2Cl2 was removed and the residue was purified by silica gel column chromatography using hexane /ethyl acetate (2/3) as eluent to get product as white solid (24.8 mg, 0.0716 mmol, 52.6%). 1H NMR (300 MHz, CDCl3): δ 7.91 (q, 2H), 7.70 (s, 1H), 7.49 (s, 1H), 7.47-7.42 (m, 2H), 4.62 (t, 2H), 3.96 (t, 2H), 3.88 (s, 3H), 3.62-3.53 (m, 6H), 3.48-3.45 (m, 2H), 1.59 (b, 1H). 13C NMR (75 MHz, CDCl3): δ 173.04, 132.76, 132.71, 130.29, 130.18, 127.71, 127.55, 124.90, 124.82, 105.99, 104.50, 72.43, 70.78, 70.41, 68.74, 61.64, 45.06, 31.22. LC/MS (ESI) m/z =347.1434 [M+H]+ and 369.1250 [M+Na]+, calc. for C18H23N2O3S =347.1429 and C18H22N2NaO3S =369.1249. Synthesis of 4. 3 (50.0 mg, 0.144 mmol), 45.4 mg triphenylphosphine (0.173 mmol), and 57.4 mg carbon tetra bromide (0.173 mmol) were added to 15 ml methylene chloride. The solution was stirred for 24 h at room temperature. Then water was added and extracted with methylene chloride. The


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organic layer was collected, and dried over anhydrous sodium sulfate. It was purified by flash chromatography

Scheme 2 Reaction of PNIS with NaOCl and the mechanism of detection.

on silica gel (EA:hexane = 1:4) afforded 4 (38.4 mg, 80%). 1 H NMR (300 MHz, CDCl3): δ 7.88 (m, 2H), 7.66 (s, 1H), 7.43 (m, 3H), 4.58 (t, 2H, J = 5.6 Hz), 3.93 (t, 2H, J = 5.4 Hz), 3.84 (s, 3H), 3.44 (m, 6H), 3.25 (t, 2H, J = 6.3 Hz); 13C NMR (75 MHz, CDCl3) δ 173.11, 132.90, 132.79, 130.36, 130.26, 127.79, 127.64, 124.97, 124.91, 106.13, 104.56, 71.25, 70.86, 70.51, 68.93, 45.21, 31.31, 30.35; LC/MS (ESI) m/z = 411.0563 [M + H]+, calc. for C18H22BrN2O2S = 410.3500. Synthesis of PNIS. 20.0 mg 4 (0.0489 mmol) and 12.8 mg triphenylphosphine (0.0587 mmol) were refluxed for 24 h in acetonitrile under nitrogen conditions. Then the reaction mixture was diluted with water and extracted with methylene chloride. The organic layer was collected, and dried over anhydrous sodium sulfate. It was purified by flash chromatography on silica gel (CH2Cl2:MeOH = 15:1) to afforded PNIS as white solid (18.6 mg, 64%). 1H NMR (300 MHz, CDCl3) : δ 7.87-7.91 (m, 2H), 7.67-7.74 (m, 9H), 7.577.63 (m, 7H), 7.52 (s, 1H), 7.40-7.44 (m, 2H), 4.50 (t, 2H, J = 5.7 Hz), 3.83-3.93 (m, 4H), 3.69-3.77 (m, 3H), 3.24 (s, 4H), 2.09 (s, 2H); 13C NMR (75 MHz, CDCl3) δ 172.98, 134.71, 134.67, 133.96, 133.82, 132.65, 132.62, 130.22, 130.15, 130.11, 129.94, 127.72, 127.62, 125.08, 125.01, 119.33, 118.18, 105.82, 104.80, 70.37, 70.12, 68.40, 64.18, 64.0,8 44.86, 31.37, 25.56, 24.86; LC/MS (ESI) m/z = 591.1947 [M + H]+, calc. for C36H36N2O2PS = 591.2235.

80% yield. Treatment of 4 with triphenylphosphine afforded the desired probe PNIS in a 64% yield. The detailed experimental procedure and characterization data are explained in the experimental section (Fig S1-9). The design strategy was based on imidazolium salt, which showed good solubility in aqueous solution, as well as photo-stability.41-43 Imidazoline-2-thione can react with OCl-, affording its imidazolium derivative,15 along with off/on fluorescence in aqueous solution (scheme 2). We firstly examined the fluorescence response of PNIS to added OCl- in PBS solution. As shown in Fig. 1a, after addition of OCl-, the fluorescence intensity of PNIS increased quickly and a new peak centered at 447 nm appeared. The response takes less than 3 min to reach stable (Fig S10). Fig. 1b shows the fluorescence intensity at 447 nm as a function of OCl- concentrations. The intensity increased greatly and reached to maximum when low concentrations of OCl- was added. The detection limit was calculated to be 2.10 × 10-7 M (Fig. S8). UV/vis absorption spectra of PNIS changed greatly after titration with OCl- (Fig. S11). The peak of 344 nm decreased and a broad peak centered around 325 nm appeared, which is consistent with the absorbance peak of naphthoimidaozlium.15 Preparative reaction of PNIS and OCl- was carried out and the mainly product was separated and determined to be compound 5 (Fig. S12).

Reaction of PNIS and OClNaOCl ( 312mL, 1mM) was added to PNIS (70 mg, 0,104 mmol) solution in CH3CN (104 mL) and stirred for 1h under room temperature. After that, solvents were removed and the residue was purified by HPLC to get 5 as a white solid (38 mg, 0.056 mmol, 53.8%). 1H NMR (300 MHz, CDCl3) : δ 10.85 (s, 1H), 8.40 (s, 1H), 8.15 (s, 1H), 8.13-8.11 (m, 1H), 8.07-8.05 (m, 1H), 7.78-7.76 (m, 4H), 7.71-7.65 (m, 11H), 7.61-7.59 (m, 2H), 4.79 (b, 2H), 4.28 (b, 3H), 3.903.84 (m, 4H), 3.76-3.75 (m, 2H), 3.36 (b, 2H), 3.29 (b, 2H). LC/MS (ESI) m/z = 280.1313 [M]2+, calc. for C36H36N2O2PS = 560.2582. Results and Discussion The synthetic route is explained in Scheme 1. Firstly, 1methyl-1H-naphtho[2,3-d]imidazole reacted with 2-(2-(2iodoethoxy)ethoxy)ethanol to give imidazolium salt 2. 2 converted to imidazoline-2-thione 3 by reaction with surfur and sodium methoxide. 3 was converted to its bromide adduct 4 using CBr4 and triphenylphosphine, in an

Figure 1 (a) Fluorescence spectra changes of PNIS (2 µM) during titrations with OCl (0-30 eq.) in PBS solution (50 mM,



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pH 7.4, DMF 0.2%). (b) Fluorescence intensity at 447 nm of PNIS (2 µM) as a function of OCl concentrations. Excitation wavelength: 325 nm, slit width: 5 nm/5 nm.

PNIS also shows high selectivity to other ROS/RNS. PNIS (2 µM) was treated with various ROS (10 µM) including OCl-, NO•, •OH, ROO•, H2O2, ONOO-, and tertbutyl hyperoxide TBHP) in PBS buffer (50 mM, pH 7.4), and there are no observable fluorescence changes except OCl-. Fig. 2 shows emission changes at 447 nm with OCl(10 μM) and an excess of other types of ROS such as ROO• (1 mM), NO• (1 mM), H2O2 (1 mM), TBHP (1 mM), ONOO(100 mM), and •OH (200 µM). It is noteworthy to emphasize that an amount more than 100 times larger than OClof other ROS did not induce any significant changes. To test the robustness of PNIS, the effects of some biological molecules such as glucose, ATP and histidine were studied, the results shows that the solution fluorescence still increase greatly (Fig. S13).

Figure 2. Fluorescence spectra of PNIS (2 µM) with various ROS (10 µM) in PBS solution (50 mM, pH 7.4, 0.2% DMF), such as OCl , NO•, •OH, ROO•, H2O2, ONOO , and TBHP. Excitation wavelength: 325 nm, slit width: 5 nm/5 nm.

Figure 3. Fluorescence intensity at 447 nm of PNIS with various ROS: OCl (10 μM), ROO• (1 mM), NO• (1 mM), H2O2 (1 mM), TBHP (1 mM), ONOO (100 mM), and •OH (200 μM). [PNIS] = 2 μM, in PBS buffer (50 mM, pH 7.4, DMF 0.2%). Excitation wavelength: 325 nm, slit width: 5 nm/5 nm.

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The fluorescence response of PNIS was also measured in two-photon (TP) processes including TP fluorescent titration, TP absorption cross section, and quadratic dependences of incident laser power and shown in Figure S14. Upon binding with OCl , TP excited fluorescence (TPEF) intensity of PNIS increased dramatically, as displayed in both titration and TP action spectra. Further, the power dependence of PNIS showed quadratic dependence of the TPEF intensity on the excitation laser power, confirming the nonlinear absorption. These results confirmed that the probe is effective TP “turnon” probe. Then we sought to apply the probe to the imag-

ing of OCl- in live cells by TPM. TPM imaging of probelabeled cells was performed with an incubating system (Chamlide IC; Live Cell Instrument) to allow for a stable cell environment through the maintenance of the proper temperature, humidity, and pH. Upon treatment with NaOCl (200 μM), TPM images of HeLa cells labeled with PNIS showed significant enhancement of the two-photon fluorescent signals (Fig. 4a and 4d), indicating the effective cell loading ability and TP turn-on response in the cells. Similar results were observed with HepG2 cells and primary cultured astrocytes (Fig. 4).

Figure 4. TPM images of (a,d) HeLa cells, astrocytes (b,e), and HepG2 cells (c,f) labeled with 10 μM PNIS (a-c) before and (d-f) after pretreatment with 200 μM NaOCl for 30 min. The TPM images were obtained by collecting the TPEF at 400-600 nm upon excitation at 700 nm with femtosecond pulses. Scale bars: (upper) 48 and (lower) 18 μm.

Figure 5. (a) Two-photon microscopy and (b) one-photon microscopy images of HeLa cells labeled with (a) PNIS, and (b) MitoTracker Red FM (MTR). (c) Merged image. Cells were pretreated with 200 μM NaOCl for 30 min before labeling with probes. The wavelengths for TP and OP excitation were 700 and 552 nm, respectively, and the corresponding emissions were collected at 400-550 nm (PNIS) and 650-700 nm (MTR). Scale bar: 19 μm.


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To demonstrate the ability of PNIS in imaging of OCl- in mitochondrial, we firstly tested fluorescence mitochondrial-localizing ability in live cells with added OCl-. We performed a colocalization experiment with a well-known mitochondrial staining dye, MitoTracker Red (MTR), through the use of TPM. As shown in Fig. 5, the TPM image of HeLa cells labeled with PNIS merged well with the one-photon microscopy image of MTR. The Pearson’s colocalization coefficient, a correlation of the intensity distribution between PNIS and MTR, was calculated as 0.83. These data support the selective imaging of OCl- in mitochondria. PNIS was then applied to the imaging of endogenous

OCl-. It is known that lipopolysaccharide (LPS), IFN-γ, and phorbol myristate acetate (PMA) can generate OCl in RAW 44

264.7 macrophages cells. More specifically, PMA generates hydrogen peroxide (H2O2), and then myeloperoxidase (MPO) - 45 converts hydrogen peroxide to OCl . MPO inhibitors such as 4-aminobenzoic acid hydrazide (ABAH) and flufenamic 46 acid (FFA) have been used. Fig. 6 shows the TPM images of RAW 264.7 cells with exogenous and endogenous OCl . A brighter image was observed in RAW 264.7 cells pretreated with OCl (Fig. 6b). The fluorescence intensity of PNIS in the cells pretreated with LPS, IFN-γ, and PMA was also enhanced (Fig. 6c). The introduction of MPO inhibitors such as ABAH (Fig. 6d) and FFA (Fig. 6e), induced a decrease in fluorescence intensity. These results support the notion that PNIS successfully images the endogenous OCl in RAW 264.7 mac-

rophages cells.

In conclusion, here, we synthesized a novel two-photon probe based on imidazoline-2-thione (PNIS) for the selective detection of mitochondrial OCl- among the various ROS. PNIS showed good selectivity for OCl- in living cells. Conversely, other ROS such as NO•, •OH, ROO•, H2O2, ONOO-, and TBHP did not significantly enhance the fluorescence. PNIS was successfully applied to the imaging of OCl- in HeLa cells. The use of PNIS to detect endogenous OCl- was further examined in RAW 264.7 macrophage cells using TPM. OCl- generation in RAW 264.7 macrophage cells, as a result of LPS, IFN-γ, and PMA, could be visualized by the fluorescence enhancement of PNIS. Conversely, the introduction of MPO inhibitors such as 4aminobenzoic acid hydrazide (ABAH) and flufenamic acid (FFA) induced a decrease in fluorescence. These outcomes demonstrate that our probe can selectively detect mitochondrial OCl- in situ.

ASSOCIATED CONTENT Supporting Information The characterizations of probe PNIS and other chemicals, time course changes of PNIS fluorescence intensity with added OCl , UV/vis spectra changes of PNIS as the titrations of OCl , fluorescence titrations of OCl with biological molecules and measurement of TP cross section are available free of charge via the Internet at http://pubs.acs.org.

AUTHOR INFORMATION Corresponding Author * Fax: (+82) 2-3277-2384 (J. Y.). E-mail: [email protected] (J. Y.) and [email protected] (H. M. K.).

Author Contributions †

These authors contributed equally.

Notes The authors declare no competing financial interest.

ACKNOWLEDGMENTS

Figure 6. TPM images of RAW 264.7 macrophage cells labeled with 10 μM PNIS. (a) Control image. (b) Cells pretreated with NaOCl (200 μM) for 30 min, and then incubated with PNIS. (c) Cells pretreated with LPS (100 ng/ml) for 16 h, IFNɣ (50 ng/ml) for 4 h, PMA (10 nM) for 30 min, and then incubated with PNIS. (d) Cells pretreated with LPS (100 ng/ml) for 16 h, IFN-ɣ (50 ng/ml) for 4 h, 4-ABAH (50 μM) for 4 h, and then incubated with PNIS. (e) Cells pretreated with LPS (100 ng/ml) for 16 h, IFN-ɣ (50 ng/ml) for 4 h, FAA (50 μM) for 4 h, and then incubated with PNIS. (f) Average TPEF intensities of (a–f). The TPM images were obtained by collecting the TPEF at 400-600 nm upon excitation at 700 nm with femtosecond pulses. Scale bars: (a,b) 19, (c,d) 20, and (e) 24 μm.

Conclusion

J. Y. acknowledges a grant from the National Creative Research Initiative program of the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (No. 2012R1A3A2048814). H. M. K. acknowledges a grant from the National Research Foundation of Korea (NRF), funded by the Korean government (MSIP) (No. 2012R1A2A1A03670456). S. N. acknowledges the National Research Council of Science and Technology through the Degree & Research Center program (DRC-14-1-KBSI). Mass spectral data were obtained at the Korean Basic Science Institute (Daegu) using a Jeol JMS 700 high-resolution mass spectrometer.

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