Studying Interfacial Reactions of Cholesterol Sulfate in an Unsaturated Phosphatidylglycerol Layer with Ozone Using Field Induced Droplet Ionization Mass Spectrometry
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Field-induced droplet ionization (FIDI) is a recently developed ionization technique that can transfer ions from the surface of microliter droplets to the gas phase intact. The air-liquid interfacial reactions of cholesterol sulfate (CholSO4) in a 1-palmitoyl-2-oleoyl-sn-phosphatidylglycerol (POPG) surfactant layer with ozone (O3) are investigated using field-induced droplet ionization mass spectrometry (FIDI-MS). Time-resolved studies of interfacial ozonolysis of CholSO4 reveal that water plays an important role in forming oxygenated products. An epoxide derivative is observed as a major product of CholSO4 oxidation in the FIDI-MS spectrum after exposure of the droplet to O3 for 5 s. The abundance of the epoxide product then decreases with continued O3 exposure as the finite number of water molecules at the air-liquid interface becomes exhausted. Competitive oxidation of CholSO4 and POPG is observed when they are present together in a lipid surfactant layer at the air-liquid interface. Competitive reactions of CholSO4 and POPG with O3 suggest that CholSO4 is present with POPG as a well-mixed interfacial layer. Compared with CholSO4 and POPG alone, the overall ozonolysis rates of both CholSO4 and POPG are reduced in a mixed layer, suggesting the double bonds of both molecules are shielded by additional hydrocarbons from one another. Molecular dynamics simulations of a monolayer comprising POPG and CholSO4 correlate well with experimental observations and provide a detailed picture of the interactions between CholSO4, lipids, and water molecules in the interfacial region.
Key wordsField induced droplet ionization Interfacial chemistry Ozonolysis Cholesterol sulfate Unsaturated phospholipid Reaction kinetics
Cholesterol sulfate (CholSO4) is a naturally occurring cholesterol (Chol) analogue widely found in various tissues [1, 2, 3, 4, 5]. Its physiologic function is only partially understood. However, CholSO4 is generally known to play roles in stabilizing and modifying the properties of cell membranes. It protects erythrocytes from osmotic lysis  and regulates sperm capacitation . A recent study using mass spectrometry has reported CholSO4 as a potential biomarker of human prostate cancer . The structure of CholSO4 is identical to Chol except for its anionic sulfate functionality. Both molecules exert comparable effects in stabilizing and modifying properties of cell membranes . However, presence or absence of the sulfate moiety also exerts dissimilar effects on cell membranes. For example, studies have shown that CholSO4 increases the order of acyl chains of phosphatidylcholine for temperatures higher than the gel-to-liquid crystal transition point, while it decreases the order for temperatures below the phase transition point [6, 7, 8]. A simulation study of interactions of 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) with CholSO4 and with Chol reveals that the hydrophobic rings of both species occupy similar locations in acyl chain bilayers . The simulation also demonstrates that the head group of CholSO4 is located slightly below the lipid phosphate group, forming more hydrogen bonds with water molecules than is the case for the hydroxyl group of Chol . This leads to stronger interactions between DPPC and CholSO4, which increases the order effect on DPPC acyl chains, but the effect is limited to adjacent DPPC.
The double bond of the oleoyl group of monounsaturated phospholipid, which is the most common unsaturated lipid found in all membranes, is located at the middle (ω-9) of the acyl chain . Previous theoretical studies have indicated that this specific location of the cis double-bond allows a maximum surface area per lipid, thus providing the highest fluidity of the lipid layer . The double-bond at ω-9 also weakens condensing and ordering effects induced by Chol [10, 11]. This effect is proposed to be caused by co-localization of the phospholipid double-bond and the hydrocarbon chain of Chol . In contrast to Chol, interactions of CholSO4 in unsaturated phospholipid layers have not been as thoroughly investigated. As reported in previous studies of a saturated phospholipid membrane, the common hydrophobic rings and tails of CholSO4 and Chol may exhibit similar interactions with acyl chains of unsaturated phospholipids, with the different head groups being the main source of distinctive behavior.
Field-induced droplet ionization mass spectrometry (FIDI-MS) employs a soft ionization technique to sample ions from the surface of microliter liquid droplets to a mass analyzer [12, 13, 14, 15]. Utilizing FIDI-MS, several studies of time-resolved heterogeneous reactions at air-liquid interfaces have been reported [14, 15]. For example, the location and orientation of SP-B1-25 (a shortened version of human surfactant protein B) in a lipid surfactant layer has been deduced from experimentally observed reactivity with ozone (O3) using a FIDI-MS methodology . In addition, detailed mechanistic studies of the heterogeneous oxidation of unsaturated phospholipids as well as the alteration of phospholipid compositions resulting from reaction with O3 at the air-liquid interface have also been reported .
2.1 Chemicals and Reagents
The sodium salt of CholSO4 is purchased from Sigma-Aldrich (St. Louis, MO, USA), 25-[N-[(7-nitro-2-1,3-benzoxadiazol-4-yl)methyl]amino]-27-norcholesterol (25-NBD Chol) and the sodium salt of POPG are purchased from Avanti Polar Lipid (Alabaster, AL, USA) and used without further purification. All solvents (water and methanol) are HPLC grade and purchased from EMD Chemicals Inc. (Gibbstown, NJ, USA).
2.2 Air-Liquid Interfacial Oxidation by O3
The FIDI-MS instrument is based on a design previously described by Grimm and Beauchamp . A stainless steel capillary with ~2 mm o.d. hanging droplet of analyte solution is located between the atmospheric sampling inlet of a mass analyzer (Thermo Finnigan LCQ Deca mass spectrometer) and a parallel plate electrode. The droplet is exposed to O3 for a desired period of time between 0 to 60 s. FIDI sampling is then achieved by applying pulsed voltages of –4 and –2 kV to the parallel plate electrode and supporting capillary, respectively. The FIDI-MS spectra reported in this study are obtained by averaging 10–30 individually acquired spectra from separately prepared droplets. Ion abundances are analyzed by measuring peak areas in FIDI-MS spectra. Approximately 20 ppm O3 is generated using a pencil-style UV calibration lamp (model 6035; Oriel). The ozone concentration is measured spectrophotometrically using an absorption cell with 10 cm path length. The ozone concentration is calculated as ~20 ppm in air with a molar absorption coefficient of 1.15 × 10–17 cm2 molecule–1 in a flow that continually bathes the droplet at ~ 1500 mL min–1. 50 μM CholSO4 or a mixture of 50 μM CholSO4 and 50 μM POPG in 1:1 (by volume) water and methanol feed the droplet source.
2.3 Solution Phase and Solid Phase O3 Reactions
A continuous flow of ~20 ppm O3 in He is bubbled into a 50 μM CholSO4 solution (200 μL) in 1:1 (by volume) water and methanol solvent for 15, 30, 45, and 60 s for solution phase O3 reactions. For solid phase reactions, a continuous flow of ~20 ppm O3 in He is applied to a dried ~1.4 × 10–4 g of CholSO4 film in 20 mL glass vial for 30 min, 2 h, and 12 h. The dried CholSO4 film is prepared by drying 300 μL of 1 mM CholSO4 solution dissolved in 2:1 (by volume) chloroform and ethanol solvent under dry N2. The film is then placed under vacuum overnight. For analysis a sample solution is prepared with a total 50 μM concentration using methanol for electrospray ionization (ESI). Product analysis is performed on a Thermo Scientific LTQ Velos dual ion trap mass spectrometer in negative ion mode.
2.4 Fluorescence Microscopy
The fluorescence labeled CholSO4 (25-NBD CholSO4) is prepared from 25-NBD cholesterol using modified method of Duff  by Sandhoff et al. . In brief, 0.355 mg of 25-NBD cholesterol is dissolved 20 μL of 5 mg/mL sulfur trioxide pyridine complex solution in absolute pyridine. After 10 min in room temperature, 2.1 μL of 314.1 mM barium sulfate is added and left for 10 min in room temperature. Then, the sample is incubated in distilled water in the refrigerator (4°C) for 1 h. Lastly, the solution is centrifuged at 10,000 rpm for 10 min at 15°C and stored at −20°C. The purity of the derived 25-NBD CholSO4 is checked using TLC and single spot is found from normal phase chromatography. Fluorescence microscopy observation of air-liquid interface is carried out using fluorescence microscope (Eclipse 80i; Nikon) with a mercury lamp as a light source. Lipid monolayer is prepared on the 300 μL water or water/methanol (1:1 by volume) droplet, which is deposited on the microscope slide with a cavity. The lipid layer composed with the mixture of 10 μM CholSO4 and 10 μM POPG contains 0.5 mol% of 25-NBD cholesterol sulfate.
2.5 Computational Modeling
Molecular dynamics (MD) simulations are performed with the all-atom CHARMM22 force field [19, 20] using the CHARMM package . Flexible TIP3P water potential is used with Hooke’s constants of 900 kcal mol–1 Å–2 for the OH bond and 110 kcal mol–1 rad–2 for the H-O-H angle. Models of CholSO4 and POPG lipid monolayer-water system consist of 48 hexagonally-packed lipids and are simulated at different surface densities of 55, 60, 65, and 70 Å2 per lipid molecule. A wall potential described by the repulsive part of V = ε[2/15(σ/r)9–(σ/r)3] with ε = 0.1521 kcal/mol and σ = 3.1538 Å is employed to prevent water molecules from diffusing out of the box. The force field parameters of CholSO4 are generated based on all-atom Chol parameter set reported by Pitman et al . The modifications are on the head group of CholSO4: the parameters of S and O atoms are directly adopted from methylsulfate parameters [23, 24] and the charge on the linking carbon on the cholesteryl ring is adjusted to the correct total charge of the entire molecule (−1). These systems are composed of a 1:3.4 CholSO4: POPG ratio and generated in two dimensions. The box dimensions of the MD simulations are (55.21 × 55.21 × 59.82 Å) for the 55 Å2/lipid case, (57.67 × 57.67 × 59.82 Å) for the 60 Å2/lipid, (60.02 × 60.02 × 59.82 Å) for the 65 Å2/lipid, and (62.28 × 62.28 × 59.82 Å) for the 70 Å2/lipid. Electrostatic and Lennard-Jones interaction were considered with 12 Å cutoffs and 10 Å tapers. The switching is performed so that the force, not the potential, smoothly decays to zero within this 2 Å tapering region. Each simulation consists of 0.5 ns equilibration at 300 K using Nose-Hoover thermostat NVT MD simulations with a relaxation time of 0.1 ps, followed by 2.0 ns of production NVT simulations for the molecular distribution analysis.
3 Results and Discussion
3.1 Air-Liquid Interfacial Reaction of CholSO4 with O3
Experimentally determined ozonolysis rate constant values of CholSO4 and POPG
CholSO4 (with POPG)
POPG (with CholSO4)
k 2 a
0.27 ± 0.05
0.18 ± 0.03
0.3708 ± 0.0002
0.30 ± 0.03
k 1 b
(5.4 ± 1.0) × 10–16
(3.6 ± 0.6) × 10–16
(7.4 ± 0.6) × 10–16
(5.9 ± 0.8) × 10–16
It is notable that the oxygenated products I, II, and III appear at the beginning of the ozonolysis (O3 exposure for 5 s). However, as the droplet is exposed to O3 for 15 s, a relatively low abundance of these products is observed in the FIDI-MS spectrum (Figure 1b). After 30 s exposure of O3 to the droplet, only a small amount of product III is observed along with highly abundant product IV and product V. The air-liquid interfacial ozonolysis of CholSO4 yields HHP (product IV) and MHP (product V) products as the most abundant products. In order to yield HHP, a primary ozonide (POZ) reacts with a water molecule (Scheme 2) [32, 33]. However, this unstable product converts to an aldehyde product through a proton transfer with a water molecule in the bulk phase . Abundant hydroperoxide products in the FIDI-MS spectra have been reported in our previous study of heterogeneous POPG ozonolysis . These products are observed in the spectra due to low water abundance at the air-liquid interface. Similarly, the abundant HHP product from CholSO4 ozonolysis is attributed to a relatively low water density in the CholSO4 interfacial layer. At the air-water interface, Chol assembles in ordered crystalline structures up to three molecular layers thick [34, 35]. Analysis using X-ray diffraction indicates that the Chol crystalline phase on the liquid surface comprises Chol monohydrate crystallites . It is expected that CholSO4 interacts with water molecules to a greater extent than Chol due to its anionic sulfate head group. In contrast to the behavior of Chol with the hydroxyl group, solvation of the anionic sulfate group may prevent CholSO4 from forming multilayer crystalline hydrates at the air-liquid interface.
No dimeric or trimeric product is observed in the FIDI-MS spectra. The study of Chol ozonolysis in a gas phase aerosol reported bound multimeric products, which are formed through aggregated gas phase clusters . The absence of multimeric product from heterogeneous ozonolysis suggests that CholSO4 molecules do not aggregate and form well-oriented layer structures on the surface of the droplet.
3.2 Solution and Solid Phase Reaction of CholSO4 with O3
All products observed from the air-liquid interfacial ozonolysis of CholSO4 (Figure 1a) are observed from the solution phase ozonolysis with different product distribution (Figure 2a). The solution phase ozonolysis of CholSO4 also yields HHP (product IV, m/z 531) and MHP (product V, m/z 545) products as the most abundant products. The significant difference compared with air-liquid interfacial ozonolysis is observed from the relative abundance of three oxygenated products (products I, II, and III). After bubbling of O3 for 60 s, product I (m/z 481) appears as the most abundant product in the ESI-MS spectrum while product III at m/z 513 is shown as the least abundant product among products I, II, and III.
The solid phase reaction yields different products except for triply oxygenated product (product III) at m/z 513 (Figure 2b). Singly and doubly oxygenated products are not observed at m/z 481 and 497, respectively, in the ESI-MS spectrum after applying O3 to a dried CholSO4 film for 12 h. As well, hydroperoxide products (m/z 531 and 545) are not observed. Instead, a diol (+34) product is observed at m/z 499. The absence of product I (epoxide), product II (dicarbonyl), and hydroperoxide (products IV and V) products from ozonolysis of the CholSO4 film implies that formation of these products requires a wet environment.
3.3 The Role of Water Molecules in Ozonolysis of CholSO4
Previous studies of the ozonolysis of Chol have reported that the epoxide product is observed only in a polar solvent [25, 36]. Formation of this product does not occur in an aprotic solvent . The epoxide product is also observed from free-radical peroxidation processes . Once O3 is dissolved in a polar solvent (i.e., water), unstable O3 rapidly forms secondary reactive oxygen species (ROS), including OH radical . This implies that the formation of an epoxide product (product I in Scheme 2) involves either an ozonolysis process aided by polar solvent or oxidation of CholSO4 with a secondary ROS . However, O3 has limited solubility in an aqueous solution, with a very low Henry’s law constant (0.011 M/atm) . This results in a very limited number of ROS in solution to produce the abundant epoxide product. This suggests that interactions of a Criegee intermediate (CI) or a primary ozonide (POZ) with solvent molecules (water or methanol) would be a responsible for the formation of product I (m/z 481 in Figures 1a and 2a) from the ozonolysis of CholSO4. The role of solvent molecules to yield the dicarbonyl product (product II in Scheme 2, m/z 497 in Figure 1a) is unclear, but dicarbonyl product is known to be highly abundant interfacial ozonolysis product of Chol and even suggested as a biomarker for ozone exposure of lung, where constant heterogeneous reactions occur [27, 28]. The carbonyl-acid product (product III) at m/z 513 is the least abundant product from solution phase ozonolysis (Figure 2a) while it is the most abundant product from solid phase ozonolysis (Figure 2b). The formation of a carbonyl-acid product does not require a solvent molecule. Thus, this product is highly abundant with a dry environment and observed in much lower yields in a wet environment.
As seen in Figure 1b, the abundance of the epoxide product (product I, m/z 481) is comparable to the carbonyl-acid product (product III, m/z 513) after the droplet is exposed to O3 for 5 s. However, its abundance decreases dramatically relative to other products after exposure of the droplet to O3 for 15 s. This indicates an important characteristic of the air-liquid interface. As discussed earlier, low water density in the CholSO4 layer is expected at the air-liquid interface. The limited number of water molecules around the double bond of CholSO4 is consumed to form HHP (product IV). This results in the low yield of the epoxide product via ozonolysis of CholSO4 at the air-liquid interface. A similar phenomenon is observed from the previous study of the ozonolysis of POPG at the air-liquid interface . The secondary ozonide of POPG (POPG-SOZ), which forms only under an anhydrous environment, starts building up after the limited water molecules are depleted around the double bond of POPG. The relatively low abundance of the product II is also a consequence of the limited number of water molecules around the double bond of CholSO4. Solution phase ozonolysis of CholSO4 shows a higher abundance of product II (m/z 497) than product III (m/z 513), while only the latter is observed under a dry environment (Figure 2). Once the CI forms from the POZ of CholSO4 (Scheme 2), after the limited water molecules are depleted, the carbonyl-acid product is preferentially formed at the air-liquid interface.
3.4 Air-Liquid Interfacial Reaction of a CholSO4 and POPG Mixture with O3
Ozonolysis rates of CholSO4 and POPG in the mixture decrease compared with when each molecule is present alone (Figure 3b). The observed rate constant values, k 1 , of CholSO4 and POPG are 3.6 × 10–16 cm3 molecule–1 s–1and 5.9 × 10–16 cm3 molecule–1 s–1, respectively, in a mixture (Table 1). The slower reaction rates of both CholSO4 and POPG suggest that CholSO4 is present with POPG as a well-mixed interfacial layer supporting the previous study of its miscibility to liquid extended layers . Our fluorescence microscopy observation of the mixture of CholSO4 (with 0.5 mol% 25-NBD CholSO4) and POPG at the air-liquid interface also supports this showing homogeneous one phase image (Figure S1 in the Supplemental Information). Overall, the POPG ozonolysis rate decreases by 20% and CholSO4 ozonolysis rate decreases by 34% in a mixed layer (Figure 3b) . As discussed earlier, air-liquid interfacial ozonolysis rate constants of both CholSO4 (5.4 × 10–16 cm3 molecule–1 s–1) and POPG (7.4 × 10–16 cm3 molecule–1 s–1) are comparable. In addition, the ozone concentration is assumed to be constant during the ozonolysis. The observed time delay for overall ozonolysis of POPG and CholSO4 implies that double bonds of POPG and CholSO4 are more shielded by additional hydrocarbons from each other at the air-liquid interface. Slightly longer delay for the ozonolysis of CholSO4 indicates that shielding effect of POPG acyl chain to the double bond of CholSO4 is slightly higher compared with the shielding effect of CholSO4 to the POPG double bond. The relative abundance of CholSO4 increases after 30 s of O3 exposure (Figure 3c). Slightly more reactive POPG yields more hydrophilic ozonolysis products, which diffuse into the aqueous droplet  increasing relative abundance of CholSO4 in the interfacial surfactant layer.
It is notable that the POPG-SOZ is formed before POPG is depleted in the spectrum. Previous study of the heterogeneous ozonolysis of POPG has shown that POPG-SOZ starts building up after POPG is depleted on the surface of the droplet . The early appearance of POPG-SOZ suggests rapid depletion of the limited number of water molecules in the hydrophobic portion of the lipid layer due to the co-consumption of water molecules with CholSO4.
3.5 Interactions of CholSO4 and POPG Double Bonds with Water Molecules at the Air-Liquid Interface
MD simulations for the mixture monolayer of CholSO4 and POPG on a water box are performed for 2.0 ns. Four different surface densities (55, 60, 65, and 70 Å2/lipid) of CholSO4 and a POPG mixture monolayer are used for the simulations. These surface densities are reported as a reasonable density range of a lipid monolayer at the air-liquid interface [41, 42, 43]. The ratio between CholSO4 and POPG in the monolayer is set to be 1:3.4. In order to investigate the effect of CholSO4 abundance in the monolayer, additional MD simulation for the mixture monolayer of CholSO4 and POPG with ratio of 1:1 is also performed for 2.0 ns using the surface density of 60 Å2/lipid.
The number of sp3 carbons per sp2 carbon calculated from the surface of surfactant layer to the locations of double bonds using a surface density of 60 Å2/lipid
1:0 (CholSO4 only)
0:1 (POPG only)
We have utilized the FIDI-MS technique to examine the effect of environmental exposures on the surfactant layer at the air-liquid interface. This study provides details for the reaction of CholSO4 with O3 to understand the unique chemistry of this molecule at an air-liquid interface. Time-resolved studies of ozonolysis of CholSO4 at the air-liquid interface reveal that a limited amount of water around double bonds of CholSO4 plays an important role in yielding oxygenated products. The epoxide and dicarbonyl products are observed only when water molecules are present around double bonds of CholSO4. Competitive oxidation of CholSO4 and POPG at the air-liquid interface suggests that both lipids form a mixed interfacial layer when they are present together in a lipid surfactant layer. In a mixed layer, the double bonds of CholSO4 and POPG are more shielded by additional hydrocarbons from each other resulting in a time delay for the ozonolysis of both molecules. MD simulations of a mixed interfacial monolayer of CholSO4 and POPG provide a detailed picture of the interactions between POPG, CholSO4, and water molecules in the interfacial region. In these simulations, the location and orientation of CholSO4 relative to POPG provide a rationalization for the experimental observations.
The authors acknowledge financial supported for this work by Basic Science Research Program (to H.I.K.; grant no. 2010–0021508) and by WCU Program (to S.M.C. and Y.M.R.; grant no. R32-2008-000-10180-0) through the National Research Foundation of Korea funded by the Ministry of Education, Science, and Technology. The authors also acknowledge financial support provided by the Beckman Institute Mass Spectrometry Resource Center and National Science Foundation of the United States under grant no. CHE-0416381 (to J.L.B., PI).
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