Coupling PaternÃ²-BÃ¼chi Reaction with Surface-Coated Probe

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Coupling Paternò-Büchi Reaction with Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for In Vivo and Microscale Profiling of Lipid C=C Location Isomers in Complex Biological Tissues Jiewei Deng, Yunyun Yang, Yaohui Liu, Ling Fang, Li Lin, and Tiangang Luan Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.8b05803 • Publication Date (Web): 05 Mar 2019 Downloaded from http://pubs.acs.org on March 11, 2019

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Analytical Chemistry

Coupling Paternò-Büchi Reaction with Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for In Vivo and Microscale Profiling of Lipid C=C Location Isomers in Complex Biological Tissues Jiewei Deng†, Yunyun Yang*,‡, Yaohui Liu‡, Ling Fang†,#, Li Lin†, and Tiangang Luan*,†,§ †State

Key Laboratory of Biocontrol, South China Sea Bio-Resource Exploitation and Utilization Collaborative Innovation Center, School of Life Sciences, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, China ‡Guangdong Engineering and Technology Research Center for Ambient Mass Spectrometry, Guangdong Provincial Key Laboratory of Emergency Test for Dangerous Chemicals, Guangdong Institute of Analysis (China National Analytical Center Guangzhou), 100 Xianlie Middle Road, Guangzhou 510070, China §School of Environmental Science and Engineering, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, China #Instrumental

Analysis & Research Center, Sun Yat-Sen University, 135 Xingangxi Road, Guangzhou 510275, China

ABSTRACT: Lipids are important structural components of biological systems, and lipid C=C locations play important roles in their biophysical and biochemical properties. Rapid, in vivo, in situ, and microscale lipidomics investigation (including precise identification of lipid C=C locations and isomers) of biological specimen has great potential for clinical diagnosis, biological studies, and biomarker discovery. Here we report a novel lipidomics methodology by coupling Paternò-Büchi (PB) reaction with surface-coated probe nanoelectrospray ionization mass spectrometry (SCP-nanoESI-MS) for in vivo, in situ, and microscale analysis of lipid species and C=C location isomers in complex biological tissues. The proposed SCP-PB-nanoESI-MS method was performed by application of a biocompatible solid-phase microextraction (SPME) probe for in vivo, in situ, and microscale sampling and extraction of lipids from biological tissues, and then some spray solvent containing PB reagent was applied to desorb lipid species enriched on SPME probe within a nanospray tip. Subsequently, ultraviolet irradiation was employed to initiate PB reaction for unsaturated lipids within the nanospray tip. After that, a high voltage was applied on the SPME probe to induce nanoESI for MS analysis under ambient and open-air conditions, and collision-induced dissociation was performed to the PB reaction product ions for determination of lipid C=C locations and isomers. By using our proposed SCP-BP-nanoESI-MS method, microscale investigation of lipid compositions and C=C location isomers for lipid droplet of Perilla seed and human intestinal tissue were successfully achieved, and in vivo analysis of lipid species and C=C locations for zebrafish was accomplished.

Lipids are important components of biological systems and play multiple roles in biological functions such as cell barriers, membrane matrices, energy storages, and signalling molecules, etc.1-3 Previous studies have demonstrated that many diseases such as atherosclerosis, Alzheimer’s disease, and even cancers were closely related with the abnormities of lipid metabolism.4, 5 The ability of lipids to perform their biophysical and biochemical roles relies on their chemical structures. Biological lipids are highly complex and very dynamic, with different aliphatic chains lengths, diverse branches, various degrees of unsaturation, and diversiform carbon-carbon double bond (C=C) locations constituting a large number of individual lipid molecular species. The C=C location is an important parameter to determine the overall shape and structure of unsaturated lipids, which is closely related to the chemical and biological function of lipid species.6 For instance, ω-3 polyunsaturated fatty acids (PUFAs) are essential substances for the functional development of brain and retina, while no such effects have been observed for ω-6 PUFAs.7, 8 In addition, the lipid bilayer properties, e.g., lateral pressure profiles, lateral membrane dynamics, and intramolecular

dynamics, are also affected by the locations of C=C bonds.9, 10 Moreover, the location changes of C=C bonds for some lipid species might relate to cancers.11 Thus, determination of the lipid C=C locations and isomers is an essential and important job for lipidomics investigation. Mass spectrometry (MS) has always been the most powerful tool for lipidomics investigation. Rich molecular information including the classes of lipids, the fatty acyl/alkyl composition, and the degree of unsaturation, etc., can be readily obtained via mass spectrometric analysis. However, accurate detection of C=C locations for unsaturated lipids and discrimination of lipid C=C isomers are difficult to achieve using conventional MS methods. Recently, Xia and Ouyang’s group reported the introduction of online Paternò-Büchi (PB) reaction with nanoelectrospray ionization (nanoESI)-MS method,11-15 by using acetone as reagent for PB reaction and subsequent collision-induced dissociation (CID) experiments, identification and quantitation of C=C location isomers for a series of unsaturated lipids such as fatty acid (FA), phosphatidylcholine (PC), and cholesteryl esters were successfully achieved. In addition, online coupling PB 1

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samples, and 3) determination of lipid C=C locations and isomers for TAGs, which is difficult to achieve using previous reported acetone/water PB reaction system.

reaction with direct infusion ESI-MS16 and ambient MS17 were also reported by their group, to achieve rapid and in situ analysis of lipid C=C location isomers for complex biological samples. Undoubtedly, these studies have enlighted a simple and rapid approach to determine lipid C=C locations and discriminate lipid C=C isomers. Although remarkable results have been achieved by the aforementioned PB reaction-based MS methods, there are still several problems need to be addressed: 1) triradylglycerols (TAGs, a class of important lipid species with low water solubility but present in biological systems with high contents) are hardly soluble in acetone/water, and their mass spectrometric analysis using acetone/water as solvent is very difficult. None of above studies has reported the determination of C=C locations and isomers for TAGs using acetone/water PB reaction system; 2) the lipid profiles for real biological samples usually cover hundreds of Da from low-to-high mass region. However, the use of acetone as PB reaction reagent results in a series of 58-Da mass increase product ions, which are easily overlapped with other original lipid species in the MS spectrum (e.g., the product ions of PCs are easily overlapped with the original ions of TAGs), increasing the difficulty in the discrimination of product ions from original ions for analysis of real biological samples; and 3) the abovementioned PB methods are still difficult to achieve in vivo and microscale lipidomics investigation. To solve these problems, here we reported a novel lipidomics method by coupling PB reaction with surfacecoated probe (SCP)-nanoESI-MS (developed in our previous studies)18, 19 for rapid, direct, in vivo, in situ, and microscale analysis of lipid species and determination of C=C location isomers. Benzophenone was introduced as the reagent for PB reaction, which allowed PB reaction to perform with desirable reaction efficiency in the solvent system suitable for MS analysis of most lipid species (including PCs and TAGs) such as methanol, methanol/water, and methanol/chloroform, etc. In addition, the use of benzophenone as PB reagent results in a 182-Da mass increase for product ions,20, 21 of which are more easily discriminated from other original ions in the MS spectrum, facilitating the identification of C=C locations and isomers for complex biological samples. The proposed SCPPB-nanoESI-MS method was performed by using a biocompatible solid-phase microextraction (SPME) probe developed in our previous study19 (possessing a probe-end diameter of less than 5 µm and showing excellent biocompatibility as well as high enrichment capacity towards lipid species) for in vivo, in situ, and microscale sampling and extraction of lipids from biological samples. After extraction, the lipids enriched on the SPME probe were desorbed in a nanospray tip using some solvent containing PB reagent. Then, an ultraviolet (UV) lamp was applied to irradiate and initiate PB reaction for unsaturated lipids within the nanospray tip. Subsequently, a high voltage was applied on the SPME probe for nanoESI-MS analysis and CID experiments under ambient and open-air conditions. Finally, identification and quantitation of lipid species and C=C location isomers were performed via the results of mass spectrometric analysis. Several breakthroughs have been achieved in our study: 1) in vivo and microscale analysis of lipid C=C locations and isomers for unsaturated lipids in complex biological samples, 2) desirable PB reaction efficiency and high electrospray ionization efficiency in new PB solvent system, which is suitable for analysis of most lipid species in real biological

EXPERIMENTAL SECTION Materials and Reagents. PC16:0/20:4(5Z,8Z,11Z,14Z), PC18:1(6Z)/18:1(6Z), and PC18:1(9Z)/18:1(9Z) were purchased from Avanti Polar Lipids, Inc (Alabaster, AL, USA). TAG16:0/18:1(9E)/18:2(9Z,12Z), TAG18:3 (6Z,9Z,12Z)/18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z), and TAG18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z) were purchased from Larodan AB (Solna, Sweden). Benzophenone was from Sigma-Aldrich (St. Louis, MO). HPLC grade of methanol, acetonitrile, and chloroform were supplied by Burdick & Jackson (Muskeg on, MI, USA). Pure water was purified by a Milli-Q water purification system (Milford, MA). Other chemicals of analytical grade were from Guangzhou Chemical Reagent Factory (Guangzhou, China). SCP-PB-nanoESI-MS Analysis. A biocompatible surfacecoated SPME probe was controlled by a three-dimensional moving manipulator with smallest microstep size of 1 μm (FuLi-Qian-Tian Opto-Electronics Technology, Beijing, China), and inserted into a precise position of samples for sampling and extraction of ~60 s. After sampling, the loaded biocompatible surface-coated SPME probe was inserted into a nanospray tip (BG12-94-4-N-20, without metallic coating, New objective Inc., MA, USA) prefilled with 1 µL of spray solvent (containing 0.36 mg/mL benzophenone, purged with nitrogen to remove oxygen) for desorption of 30 s. Then, PB reaction was performed under the irradiation of a UV lamp (Cnlight Optoelectronic Technology Co., Ltd, Guangdong, China) with a wavelength of 254 nm placed 1.0 cm away from the nanospray tip for ~1 min. To ensure the solvent of PB reaction system can receive well-distributed and sufficient irradiation energy, the nanospray tip and the inserted SPME probe were rotated slowly during ultraviolet irradiation process. After that, the SPME probe and nanospray tip were mounted onto a 3-dimensional moving stage (Beijing Zolix instruments Co., Ltd, Beijing, China) and placed pointing to the MS inlet, adjusting to a position of 5 mm away from the MS inlet, and a high voltage of 2.5-3.0 kV was applied on the SPME probe for nanoESI-MS analysis under ambient and open-air conditions. Mass spectra were recorded using a Sarix X 7T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer (Bruker Daltonics, Bremen, Germany) or a linear trap quadrupole (LTQ) Orbitrap Elite mass spectrometer (Thermo Fisher Scientific, San Jose, CA) with an appropriate m/z range. Accurate mass measurement was accomplished by FTICR-MS with a 4M recording mode (mass resolution of ~200,000 for lipid species) or by LTQ-Orbitrap-MS with a 120,000 mass resolution mode. CID experiments were performed by FTICRMS or LTQ-Orbitrap-MS, with an appropriate isolation window and collision energy for each selected precursor ion.

RESULTS AND DISCUSSION Procedures for SCP-PB-nanoESI-MS Analysis. The schematic diagram of SCP-PB-nanoESI-MS method was shown in Figure 1, and its general steps include: 1) application of a biocompatible SPME probe for microscale sampling and extraction of lipid species from biological samples; 2) employment of some solvents containing PB reagents for desorption of lipids from the loaded biocompatible SPME 2


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Figure 1. Schematic diagrams for development of a SCP-PB-nanoESI-MS method. a) A biocompatible surface-coated SPME probe toward lipids was applied for in situ and microscale sampling and extraction of lipids from lipid droplet or biological tissue. b) Desorption, PB reaction, and MS detection. c) MS spectrum for unsaturated lipid with PB reaction. d) CID spectrum for determination of lipid C=C locations and isomers.

pairs were produced. Due to a mass difference of 150 Da between “O” and “C13H10,” two fragment ions with a 150-Da mass difference were observed in the MS/MS spectrum (Figure 1d), which were the diagnostic ions and applied to identify C=C locations. Identification of C=C Locations for Unsaturated Lipids. A representative PC specie, i.e., PC16:0/20:4(5Z,8Z,11Z,14Z) was applied for illustration. Without PB reaction, its MS spectrum was dominated by the [M+H]+ (m/z 782.5681) signal, and the signal of [M+Na]+ (m/z 804.5523) also showed certain abundant intensity (Figure 2a). When UV was irradiated for PB reaction, the benzophenone (BP) additive ions of [M+H]+ (m/z 964.6425) was generated obviously after ~0.2 min, and its intensity was increased with the increasing irradiation time and reached equilibrium at ~1 min. The intensity of [M+BP+H]+ was 30-40% of its original ion, and the signals of [M+BP+Na]+ and [M+BP+K]+ were also observed, but their intensities were much lower than that of [M+BP+H]+ (Figure 2b). Thus, [M+BP+H]+ (m/z 964.6425) was the most suitable and desirable ion for subsequent C=C location determination. CID experiment was performed to the ion, and the obtained MS/MS spectrum was shown in Figure 2c. The fragmental ion with highest abundant intensity at m/z 946.6291 was ascribed to the neutral loss of H2O (18.0106 Da) from [M+BP+H]+, and the fragmental ion with second abundant intensity at m/z 184.0729 was corresponded to the signal of phosphorycholine (C5H15NO4P+). Two fragmental ions with certain abundant intensities at m/z 526.3288 and 478.3384 were observed, which were ascribed as the neutral loss of [FA16:0 (C36H32O2, 256.2402 Da)+BP] and [FA20:4 (C20H32O2, 304.2402 Da)+BP] from [M+BP+H]+, respectively. Thus, fatty acid chains of FA16:0 and FA20:4 were contained in this substance. Besides these, four pairs of fragmental diagnostic ions with a mass difference of 150 Da (i.e., m/z 714.3785/864.5549, 674.4399/ 824.5241, 634.4092/784.4927, and 594.3785/744.4615) were observed with high abundances in the spectrum, which could be deduced that four C=C bonds were located at Δ5, Δ8, Δ11, and Δ14 of FA20:4 chain. TAG16:0/18:1(9E)/18:2(9Z,12Z) was also analyzed. Without PB reaction, its signals of [M+H]+, [M+NH4]+, [M+Na]+, and [M+K]+ were observed in the MS spectrum, and [M+Na]+ showed the most abundant intensity (Figure 2d). After UV irradiation, signals of PB product ions (i.e., [M+BP+H]+, [M+BP+NH4]+, [M+BP+Na]+, and [M+BP+K]+) were generated. In general, the signal intensity of

probe within a nanospray tip; 3) using UV irradiation to perform PB reaction for unsaturated lipid species within the nanospray tip; 4) applying a high voltage on the SPME probe to induce nanoESI for MS analysis under ambient and openair conditions, and 5) CID experiments to the PB reaction product ions for accurate detection of C=C locations for unsaturated lipids and discrimination of lipid C=C isomers. The biocompatible SPME probe was developed in our previous study,19 which showed favorable biocompatibility and high enrichment ability towards lipid species. In addition, the biocompatible SPME probe processed a tip-end diameter of less than 5 µm, which allowed it for in vivo, in situ, and microscale sampling of biological samples (Figure 1a). The sampling and extraction time was 60 s, which has been optimized in our previous study. Then, some spray solvent containing PB reagent was filled/siphoned into a nanospray tip, and the loaded biocompatible SPME probe was inserted into the nanospray tip for desorption of lipid species. Our previous study has demonstrated that methanol/chloroform (v/v=9:1) was the most desirable solvent for desorption of lipid species loaded on the SPME probe, and the desorption time was 30 s.19 In addition, methanol/chloroform (v/v=9:1) also showed high ionization efficiency for subsequent nanoESI-MS analysis. In this study, benzophenone was selected as the PB reactant, adding into the spray solvent with a concentration of 0.36 mg/mL. A previous study has demonstrated the most suitable concentration of benzophenone for PB-ESI-MS analysis of real biological samples was 2 mmol/L (~0.36 mg/mL).21 In this study, such a concentration of benzophenone gave a desirable PB reaction efficiency and had little negative effect for nanoESI-MS analysis. After desorption, an UV light with wavelength of 254 nm and power of 25 W was applied to irradiate the solvent for a time of ~60 s (similar to the previous experimental conditions),21 and PB reaction was performed for unsaturated lipid species within the nanospray tip. Then, a high voltage of 2.5-3.0 kV was applied on the SPME probe to induce nanoESI for mass spectrometric analysis (Figure 1b). PB reaction is a classic [2+2] photochemical reaction.22 In this study, C=C bonds in unsaturated lipids and carbonyl in benzophenone underwent a cycloaddition reaction, and reaction products with a 182-Da (C13H10O) mass increase from the original unsaturated lipids were generated (Figure 1c). The PB reaction product ions were well separated with their reactant ions in the mass spectrum. When CID experiments were performed to the PB product ions, C=C diagnostic ion 3



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Figure 2. SCP-PB-nanoESI-MS spectra of PC 16:0/20:4(5Z,8Z,11Z,14Z) and TAG 16:0/18:1(9E)/18:2(9Z,12Z) a, d) without and b, e) with PB reaction, and c, f) CID spectra of the PB products, respectively.

an important and challenging job in lipidomics investigation, and our proposed SCP-PB-nanoESI-MS method shows desirable performance in the relative quantitation of lipid C=C location isomers. As for PC18:1(6Z)/18:1(6Z) and PC18:1(9Z) /18:1(9Z), two isomers showed the same signals of [M+H]+ (m/z 786.60) and [M+BP+H]+ (m/z 968.68) in their MS spectra (Figures S-1a-b in the Supporting Information), and thus they could not be discriminated without CID experiments. Via CID experiment, a pair of diagnostic ions with a mass difference of 150 Da (i.e., m/z 634.4071/784.4905 were observed in the CID spectrum of PC18:1(6Z)/18:1(6Z) (Figures S-1c in the Supporting Information), which was corresponded to the C=C location at Δ6 of FA18:1. While a pair of diagnostic ions with a mass difference of 150 Da atm/z 676.4580/826.5427 (corresponding to the C=C location at Δ9 of FA18:1) were observed in the CID spectrum of PC18:1(9Z)/18:1(9Z) (Figures S-1d in the Supporting Information). Quantification of the relative contents of the two isomers was investigated, by using a series of mixtures of PC18:1(6Z)/18:1(6Z) and PC18:1(9Z)/18:1(9Z) (with the total concentration kept constant at 50 µg/mL) for analysis. The diagnostic ions from both isomers were observed in the obtained spectra (Figures S1e-g in the Supporting Information). The total ion intensities of the diagnostic ions from PC18:1(6Z)/18:1(6Z) and PC18:1(9Z)/18:1(9Z) were summed, respectively, and the ratios of their summed ion intensities (IPC18:1(6Z)/18:1(6Z)/ IPC18:1(9Z)/18:1(9Z)) were plotted against their concentrations (CPC18:1(6Z)/18:1(6Z)/CPC18:1(9Z)/18:1(9Z)). A good linearity was obtained within a wide dynamic range of concentration ratios from 1:16 to 16:1, with correlation coefficient (r) of 0.9985 (Figure S-1h in the Supporting Information). Analogously, quantitation of the relative contents for TAGs was investigated, using two representative isomers, i.e.,

[M+BP+Na]+ was most desirable, with approximately 30-40% of its original ion (Figure 2e). CID experiment was performed to the PB product ion of [M+BP+Na]+ at m/z 1061.8162, and the obtained MS/MS spectrum was shown in Figure 2f. The fragmental ions with abundant intensities at m/z 805.5750, 781.5754, and 779.5599 were corresponded to the neutral loss of C36H32O2 (256.2402 Da, FA 16:0), C18H32O2 (280.2402 Da, FA 18:2), and C18H34O2 (282.2559 Da, FA 18:1) from [M+BP+Na]+, respectively, and the fragment ions with certain intensities at m/z 623.5022, 599.5023, and 597.4867 were ascribed as the neutral loss of [FA16:0+BP], [FA18:2+BP], and [FA18:1+BP] from [M+BP+Na]+, respectively. Thus, fatty acid chains of FA16:0, FA18:2, and FA18:1 were contained in this substance. These fragmental ions make up three pairs of ions with a mass difference of 182 Da (i.e., m/z 623.5022/805.5750, 599.5023/781.5754, and 597.4867/ 779.5599), and they are very useful for the identification of length and unsaturation degree of fatty acid chains in TAG. In addition, three pairs of diagnostic ions with a mass difference of 150 Da were observed obviously (i.e., m/z 811.6425/ 961.7256, 771.6111/921.6946, and 769.5965/919.6799). The diagnostic ions of m/z 769.5965/919.6799 suggested the C=C location at Δ9 of FA18:1, and the diagnostic ions of m/z 771.6111/921.6946 and 811.6425/961.7256 suggested the C=C locations at Δ9 and Δ12 of FA18:2, respectively. It should be noted that CID experiments to the ions of [M+BP+H]+, [M+BP+NH4]+, and [M+BP+K]+ can also obtain diagnostic ion pairs with a mass difference of 150 Da to identify C=C locations for TAGs. While in most cases, the intensity of [M+BP+H]+ was relatively low, and the fragment diagnostic ion pairs from [M+BP+NH4]+ and [M+BP+K]+ were not as stable and sensitive as that from [M+BP+Na]+. Thus, CID experiment to [M+BP+Na]+ was the first choice for identification of C=C locations for TAGs. Relative Quantitation for Lipid C=C Location Isomers. Quantitation of C=C location isomers for unsaturated lipids is 4


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Analytical Chemistry nanoESI-MS shows the merits of in situ and microscale sampling as well as much less sample consumption. After PB reaction, a series of PB product ions at m/z 10501150 appeared, and these ions were well separated with their original ions (Figure 3b). CID experiments was performed to the product ions of [M+BP+Na]+ for identification of lipid C=C locations. For instance, the [M+BP+Na]+ of TAG54:9 was detected at m/z 1077.7531, and its CID spectrum was shown in Figure S-5a (Supporting Information). A pair of fragmental ions with a mass difference of 182 Da at m/z 799.5262 and 617.4538 were observed in the spectrum, which were ascribed to the neutral loss of FA18:3 (C18H30O2, 278.2246 Da) and [FA18:3+BP] from [TAG54:9+BP+Na]+, respectively. No other fragmental ions with a mass difference of 182 Da was observed, and thus only the fatty acid chain of FA18:3 was contained in this ion. In addition, three pairs of fragmental diagnostic ions with a mass difference of 150 Da (i.e., m/z 939.6465/789.5634, 979.6784/829.5965, and 1019.7116/869.6265) were observed in the CID spectrum, which were corresponded to the C=C bonds located at Δ9, Δ12, and Δ15 of FA 18:3, respectively. Thus, the ion at m/z 895.6809 was identified as TAG18:3(Δ9,12,15)_18:3(Δ9,12, 15)_18:3(Δ9,12,15), and its CID spectrum was also confirmed with that of reference substance. Analogously, the ion at m/z 897.6959 was identified as TAG18:2(Δ9,12)_18:3 (Δ9,12,15)_18:3(Δ9,12,15). Some ions are the mixture of two lipid isomers. For instance, the ion with second abundant intensity at m/z 899.7111 was calculated as [TAG54:7+Na]+ via LIPID MAPS. CID experiment was performed to its PB product ion of [TAG54:7+BP+Na]+ (m/z 1081.7842), and the obtained spectrum (Figure S-5b in the Supporting Information) demonstrated that there were three pairs of fragmental ions with a mass difference of 182 Da, i.e., m/z 799.5254/617.4541, 801.5421/619.4685, and 803.5575/621.4843. The ions of m/z 799.5254, 801.5421, and 803.5575 were ascribed to the neutral loss of FA18:1, FA18:2, and FA18:3 from [TAG54:7+BP+Na]+, respectively, and the ions at m/z 617.4541, 619.4685, and 621.4843 were ascribed as the neutral loss of [FA18:1+BP], [FA18:2+BP], and [FA18:3+BP] from [TAG54:7+BP+Na]+, respectively. Thus, three fatty acid chains of FA18:1, FA18:2, and FA18:3 were contained in this ion. Because the degree of unsaturation is 7, suggesting the possible presence of isomers at TAG18:1_18:3_18:3 and TAG18:2_18:2_18:3. In addition, six pairs of fragmental diagnostic ions with a mass difference of 150 Da (i.e., m/z 939.6489/789.5631, 941.6621/791.5817, 943.6779/793.5934, 981.6935/831.6001, 983.7087/833.6298, and 1023.7396/ 873.6567) were observed obviously in the CID spectrum. The diagnostic ion pair of m/z 939.6489/789.5631 suggested the C=C bond located at Δ9 of FA18:1, the diagnostic ion pairs of m/z 941.6621/791.5817 and 981.6935/831.6001 suggested the C=C bonds located at Δ9 and Δ12 of FA18:2, respectively, and the diagnostic ion pairs of m/z 943.6779/793.5934, 983.7087/833.6298, and 1023.7396/873.6567 suggested the C=C bonds located at Δ9, Δ12, and Δ15 of FA18:3, respectively. Thus, the ion at m/z 899.7105 was identified as the TAG18:1(Δ9)_18:3(Δ9,12,15)_18:3(Δ9,12,15) and TAG18:2(Δ9,12)_18:2(Δ9,12)_18:3(Δ9,12,15) mixtures. There are also signals derived from the mixture of three isomers. For instance, the ion at m/z 901.7274, which was calculated as [TAG54:6+Na]+ via the LIPID MAPS searching result. Upon CID to its product ion of [TAG54:6+BP+Na]+ at

TAG18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z) and TAG18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z) as an example. The MS spectra of the two isomers were very similar, with abundant intensities of [M+Na]+ (m/z 895.68) and [M+BP+Na]+ (m/z 1077.75) were observed (Figures S-2ab in the Supporting Information). CID experiments were performed to the ion of [M+BP+Na]+ for both substances. Three pairs of diagnostic ions with a mass difference of 150 Da (i.e., m/z 789.5638/939.6469, 829.5948/979.6778, and 869.6259/1019.7090) were observed evidently in the CID spectrum of TAG18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)/18:3 (9Z,12Z,15Z) (Figures S-2c in the Supporting Information), which were corresponded to the C=C locations at Δ9, Δ12, and Δ15 of FA18:3. While in the CID spectrum of TAG18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z) (Figures S-2d in the Supporting Information), there were also three pairs of diagnostic ions with a mass difference of 150 Da, but their m/z values were different. The diagnostic ions at m/z 747.5170/897.6003, 787.5478/937.6313, and 827.5787/ 977.6623 were corresponded to the C=C locations at Δ6, Δ9, and Δ12 of FA18:3. A series of mixtures of the two isomers with concentration ratios from 1:16 to 16:1 (with the total concentration kept constant at 200 µg/mL) were analyzed, and the above six pairs of diagnostic ions with a mass difference of 150 Da were observed in the obtained spectra (Figures S-2e-g in the Supporting Information). The total ion intensities of the diagnostic ions from TAG18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15 Z)/18:3(9Z,12Z,15Z) and TAG18:3(6Z,9Z,12Z)/18:3(6Z,9Z, 12Z)/18:3(6Z,9Z,12Z) were summed and used for their relative quantitation, and a good linearity (r=0.9993) was obtained with the fitting of ITAG18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z) against /18:3(9Z,12Z,15Z)/ITAG18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z)/18:3(6Z,9Z,12Z) CTAG18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)/18:3(9Z,12Z,15Z)/CTAG18:3(6Z,9Z,12Z)/18:3(6Z, 9Z,12Z)/18:3(6Z,9Z,12Z) (Figure S-2h in the Supporting Information). Microscale Lipidomics for Lipid Droplet of Perilla Seed. The developed SCP-PB-nanoESI-MS method was applied for microscale lipidomics investigation and determination of lipid C=C locations for real biological samples. A lipid droplet of Perilla seed was sampled by a biocompatible SPME probe (Figure S-3 in the Supporting Information) and then analyzed by SCP-PB-nanoESI-MS, and a series of TAG signals at m/z 850-950 were observed in the obtained spectrum without PB reaction (Figure 3a). These TAG signals were preliminarily identified via the database of LIPID MAPS using their accurate mass measurement results. For instance, the signal with highest intensity detected at m/z 895.6809 was identified as [TAG54:9+Na]+ (C57H92O6Na) based on the LIPID MAPS searching result, and the ion with second abundant intensity at m/z 899.7111 was calculated as [TAG54:7+Na]+. Our proposed SCP-nanoESI-MS method was also compared with the classical extraction and analytical methods to demonstrate its feasibility and practicability. Some Perilla seeds were extracted with methanol/chloroform (v/v=9:1) and analyzed using the conventional ESI-MS method, and the obtained spectra were shown in Figure S-4 (Supporting Information). Similar lipid profile at m/z 850-950 was observed, and the relative signal intensities were also similar, which supported the feasibility and effectiveness of lipidomics investigation using SCP-PB-nanoESI-MS method. In addition, SCP-PB5



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Figure 3. SCP-PB-nanoESI-MS spectra of a) lipid species and b) PB product ions from lipid droplet of Perilla seed.

m/z 1083.7997 (Figure S5-c in the Supporting Information), four pairs of fragmental ions with a mass difference of 182 Da (i.e., m/z 799.5261/617.4544, 801.5424/619.4681, 803.5572/ 621.4851, and 805.5572/623.5001) were emerged, which demonstrated the presence of fatty acid chains of FA18:0, FA18:1, FA18:2, and FA18:3. Because the degree of unsaturation is 6, suggesting that it was the mixture of TAG18:2_18:2_18:2, TAG18:1_18:2_18:3, and TAG18:0_18:3_18:3. Six pairs of fragmental diagnostic ions with a mass difference of 150 Da were observed. The diagnostic ion pair of m/z 941.6626/791.5823 was ascribed as the C=C bond located at Δ9 of FA18:1, the diagnostic ion pairs of m/z 943.6781/793.5931 and 983.7091/833.6298 were deduced as the C=C bonds located at Δ9 and Δ12 of FA18:2, respectively, and the diagnostic ion pairs of m/z 945.6932/795.6104, 985.7248/835.6412, and 1025.7561/ 875.6726 were corresponded to the C=C bonds located at Δ9, Δ12, and Δ15 of FA18:3, respectively. Thus, the signal of m/z 901.7274 was derived from TAG18:2(Δ9,12)_18:2(Δ9,12)_ 18:2(Δ9,12), TAG18:1(Δ9)_18:2(Δ9,12)_18:3(Δ9,12,15), and TAG18:0_18:3(Δ9,12,15)_18:3(Δ9,12,15). Other signals of TAGs were identified using the same strategy, and the results were summarized in Table S-1 (Supporting Information). In Situ and Microscale Lipid Analysis of C=C Location Isomers for Human Intestinal Tissue. A small grain of human intestinal tissue (with size of approximately 2 mm × 2 mm) after surgery (provided by Sun Yat-sen University Cancer Center) was microsampled by a biocompatible SPME probe, and then analyzed by the proposed SCP-PB-nanoESIMS method. Abundant lipid signals at m/z 840-980 were observed in the mass spectrum without PB reaction (Figure 4a), and most of them were identified as unsaturated TAGs via LIPID MAPS using the high-resolution accurate mass measurement results. Among them, signals of [TAG50:2+Na]+ (m/z 853.7268), [TAG52:3+Na]+ (m/z 879.7432), + [TAG52:2+Na] (m/z 881.7598), [TAG52:3+K]+ (m/z 895.7172), [TAG52:2+K]+ (m/z 897.7306), [TAG54:4+Na]+ (m/z 905.7586), [TAG54:3+Na]+ (m/z 907.7745), + [TAG54:2+Na] (m/z 909.7908), [TAG54:4+K]+ (m/z 921.7324), [TAG54:3+K]+ (m/z 923.7475), [TAG56:2+Na]+ (m/z 937.8176), and [TAG56:2+K]+ (m/z 953.7899), etc., were dominantly observed, and the signal of [TAG52:2+Na]+ (m/z 881.7598) showed most abundant intensity.

Figure 4. SCP-PB-nanoESI-MS spectra of a) lipid profile and b) PB product ions of dominant unsaturated lipids from human intestinal tissue. CID spectra of c) [TAG52:2+BP+Na]+ at m/z 1063.8325, and d) [TAG54:3+BP+Na]+ at m/z 1089.8431.

After PB reaction, a series of product ions with a 182-Da mass increase were observed clearly at m/z 1020-1160 of the mass spectrum (Figure 4b). These PB reaction product ions were well separated with their reactant lipid ions. The signals of [M+BP+Na]+ showed the most desirable intensities, with approximately 30% of their original ions, and thus they were selected for CID experiments to identify C=C locations and isomers. As for the highest intensity of [TAG52:2+Na]+ at m/z 881.7598, its PB product ion of [TAG52:2+BP+Na]+ at m/z 1063.8294 also showed highest intensity among the signals of PB product ions. Upon CID to this ion, and the obtained MS/MS spectrum was displayed in Figure 4c. Two pairs of fragmental ions with a mass difference of 182 Da (i.e., m/z 807.5921/625.5186 and 781.5768/599.5027) were observed. Ions at m/z 807.5921 and 781.5768 were ascribed to the neutral loss of FA16:0 and FA18:1 from [TAG52:2+BP+Na]+, respectively, and ions at m/z 625.5186 and 599.5027 were ascribed as the neutral loss of [FA16:0+BP] and [FA18:1+BP] from [TAG52:2+BP+Na]+, respectively. In addition, three pairs of fragmental diagnostic ions with a mass difference of 150 Da (i.e., m/z 921.6965/771.6118, 949.7284/799.6457, and 963.7398/ 813.6559) were observed in the MS/MS spectrum, which were corresponded to the C=C located at Δ9, Δ11, and Δ12 of FA 18:1, respectively, indicating the true C=C isomers presented in this ion, and their relative ratios were calculated to be approximately 1:0.05:0.07 via diagnostic ion intensities. Apparently, most of the FA18:1 C=C bonds present in the 6


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human intestinal tissue was located at Δ9, while low abundance isomers with C=C located at Δ11 and Δ12 were also found. A previous study has confirmed that FA18:1 Δ9 and Δ11 isomers were detected in human brain,23 and the existence of FA and TAG C=C isomers including C18:1 Δ9, Δ11 and Δ12 in human milk was also reported.24 From the information of CID experiment, we can deduced that TAG52:2 contained fatty acid chains of FA16:0, FA18:1(Δ9), FA18:1(Δ11), and FA18:1(Δ12), and it was the possible mixture of TAG16:0_18:1(Δ9)_18:1(Δ9), TAG16:0_18:1(Δ11) _18:1(Δ11), TAG16:0_18:1(Δ12)_18:1(Δ12), TAG16:0_18:1 (Δ9)_18:1(Δ11), TAG16:0_18:1(Δ9)_18:1(Δ12), and TAG16:0_18:1(Δ11)_18:1(Δ12). Analogously, the ion with second abundant intensity at m/z 907.7745 was calculated as [TAG54:3+Na]+. Upon CID to its PB product ion of [TAG54:3+BP+Na]+ at m/z 1089.8443, and the obtained spectrum was shown in Figure 4d. Only a pair of fragmental ions with a mass difference of 182 Da at m/z 807.5902/625.5176 was observed in the spectrum, which were ascribed to the neutral loss of FA18:1 and [FA18:1+BP] from [TAG54:3+BP+Na]+, respectively, and thus only the fatty acid chain of FA18:1 was contained in this ion. Three pairs of fragmental diagnostic ions with a mass difference of 150 Da (i.e., m/z 947.7106/797.6265, 975.7423784/825.6590, and 989.7542/839.6472) were observed in the CID spectrum, which were corresponded to the C=C bonds located at Δ9, Δ11, and Δ12 of FA 18:1, respectively. Thus, the ion at m/z 907.7745 was identified as the possible mixture of TAG18:1(Δ9)_18:1(Δ9)_18:1(Δ9), TAG18:1(Δ11)_18:1(Δ11) _18:1(Δ11), TAG18:1(Δ12)_18:1(Δ12)_18:1(Δ12), TAG18:1(Δ9)_18:1(Δ11)_18:1(Δ11), TAG18:1(Δ9)_18:1(Δ11) _18:1(Δ12), TAG18:1(Δ9)_18:1(Δ12)_18:1(Δ12), TAG18:1(Δ11)_18:1(Δ11)_18:1(Δ12), and TAG18:1(Δ11)_ 18:1(Δ12)_18:1(Δ12). Other signals were identified using the same strategy, and the results were summarized in Table S-2 (Supporting Information). The experimental result supported the feasibility of clinical analysis by using SCP-PB-nanoESIMS method for lipidomics investigation and identification of true C=C location isomers. In Vivo Analysis of Lipid C=C Locations for Zebrafish. In vivo analysis of lipid C=C locations for zebrafish was performed, by sampling its back muscle for SCP-PB-nanoESIMS analysis. Abundant lipid signals at m/z 750−1000 were observed in the obtained mass spectrum without PB reaction (Figure 5a), and the LIPID MAPS searching results demonstrated that most of the detected lipid species were PCs and TAGs (Table S-3 in the Supporting Information). Among them, signals of [PC32:0+K]+ (m/z 772.5251), [PC34:1+K]+ (m/z 798.5406), [PC(O-38):6+Na]+ (m/z 814.5718), [PC(O38):6+K]+ (m/z 830.5461), [PC38:6+K]+ (m/z 844.5255), [PC38:4+K]+ (m/z 848.5559), [TAG50:2+K]+ (m/z 869.6991), [TAG52:3+K]+ (m/z 895.7152), [PC44:12+K]+ (m/z 916.5248), [TAG54:4+K]+ (m/z 921.7308), [TAG56:7+K]+ (m/z 943.7158), and [TAG58:8+K]+ (m/z 969.7315), etc., were dominantly observed. After PB reaction, the product ions of dominant unsaturated lipids appeared at m/z 950-1200, with intensities of approximately 10-20% of their original ions (Figure 5b). In this case, signals of [M+BP+K]+ were predominantly observed, and signal intensities of [M+BP+Na]+ were approximately one third of those of [M+BP+K]+. It should be noted that both [M+BP+Na]+ and [M+BP+K]+ can be applied for CID experiment to identify lipid C=C locations in this case. For

Figure 5. SCP-PB-nanoESI-MS spectra of a) lipid profile and b) PB product ions of dominant unsaturated lipids obtained by in vivo analyzing zebrafish muscle, c) CID spectrum of [TAG52:3+BP+K]+ at m/z 1077.7876.

instance, the highest signal detected at m/z 895.7152 was identified as [TAG52:3+K]+ based on the LIPID MAPS searching result. After PB reaction, the signal of [TAG52:3+BP+K]+ was detected at m/z 1077.7876, and its CID spectrum was shown in Figure 5c. The fragmental ions at m/z 793.5193, 795.5348, 797.5500, 799.5589, 821.5484, and 823.5656 were corresponded to the neutral loss of FA18:0, FA18:1, FA18:2, FA18:3, FA16:0, and FA16:1 from [TAG52:3+BP+K]+, respectively, and the fragment ions at m/z 611.4465, 613.4519, 615.4866, 617.4857, 639.4779, and 641.4931 were ascribed as the neutral loss of [FA18:0+BP], [FA18:1+BP], [FA18:2+BP], [FA18:3+BP], [FA16:0+BP], and [FA16:1+BP] from [TAG52:3+BP+K]+, respectively. Thus, fatty acid chains of FA16:0, FA16:1, FA18:0, FA18:1, FA18:2, and FA18:3 were contained in this ion. Seven pairs of diagnostic ions with a mass difference of 150 Da were observed obviously. The diagnostic ion pair of 963.6801/813.6016 was ascribed as the C=C bond located at Δ9 of FA16:1, the diagnostic ion pair of 935.6495/785.5715 was ascribed as the C=C bond located at Δ9 of FA18:1, the diagnostic ion pairs of 937.6676/787.5829 and 977.7119/827.6176 were deduced as the C=C bonds located at Δ9 and Δ12 of FA18:2, respectively, and the diagnostic ion pairs of 939.6808/789.5958, 979.7119/829.6268, and 1019.7439/869.5676 were corresponded to the C=C bonds located at Δ9, Δ12, and Δ15 of FA18:3, respectively. Thus, the signal of m/z 895.7152 was derived from TAG16:0_18:1(Δ9)_18:2(Δ9,12), TAG16:0_18:0_18:3(Δ9,12, 15), TAG16:1(Δ9)_18:1(Δ9)_18:1(Δ9), and TAG16:1(Δ9)_ 18:0_18:2(Δ9,12). Analogously, other signals were identified and summarized in Table S-3 (Supporting Information).

CONCLUSION In summary, we have demonstrated the development of a novel SCP-PB-nanoESI-MS method and its application for in vivo and microscale profiling of lipid C=C location isomers for complex biological tissues. Several breakthroughs have been achieved in our study: 1) in vivo and microscale analysis 7



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Time-of-Flight Secondary Ion Mass Spectrometry. Anal. Chem. 2017, 90, 1072-1076. (4) Li, M.; Yang, L.; Bai, Y.; Liu, H., Analytical Methods in Lipidomics and Their Applications. Anal. Chem. 2014, 86, 161175. (5) Bang, G.; Kim, Y. H.; Yoon, J.; Yu, Y. J.; Chung, S.; Kim, J. A., On-Chip Lipid Extraction Using Superabsorbent Polymers for Mass Spectrometry. Anal. Chem. 2017, 89, 13365-13373. (6) Martinez-Seara, H.; Róg, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M.; Reigada, R., Interplay of Unsaturated Phospholipids and Cholesterol in Membranes: Effect of the Double-Bond Position. Biophys. J. 2008, 95, 3295-3305. (7) Uauy, R.; Peirano, P.; Hoffman, D.; Mena, P.; Birch, D.; Birch, E., Role of Essential Fatty Acids in the Function of the Developing Nervous System. Lipids 1996, 31, S167-S176. (8) Uauy, R.; Mena, P.; Rojas, C., Essential Fatty Acids in Early Life: Structural and Functional Role. Proc. Nutr. Soc. 2000, 59, 315. (9) Ollila, S.; Hyvönen, M. T.; Vattulainen, I., Polyunsaturation in Lipid Membranes: Dynamic Properties and Lateral Pressure Profiles. J. Phys. Chem. B 2007, 111, 3139-3150. (10) Martinez-Seara, H.; Róg, T.; Pasenkiewicz-Gierula, M.; Vattulainen, I.; Karttunen, M.; Reigada, R., Effect of Double Bond Position on Lipid Bilayer Properties: Insight Through Atomistic Simulations. J. Phys. Chem. B 2007, 111, 11162-11168. (11) Ma, X.; Chong, L.; Tian, R.; Shi, R.; Hu, T. Y.; Ouyang, Z.; Xia, Y., Identification and Quantitation of Lipid C=C Location Isomers: A Shotgun Lipidomics Approach Enabled by Photochemical Reaction. Proc. Natl. Acad. Sci. USA 2016, 113, 2573-2578. (12) Ma, X.; Xia, Y., Pinpointing Double Bonds in Lipids by Paternò-Büchi Reactions and Mass Spectrometry. Angew. Chem. Int. Ed. 2014, 53, 2592-2596. (13) Ma, X.; Zhao, X.; Li, J.; Zhang, W.; Cheng, J.X.; Ouyang, Z.; Xia, Y., Photochemical Tagging for Quantitation of Unsaturated Fatty Acids by Mass Spectrometry. Anal. Chem. 2016, 88, 89318935. (14) Ren, J.; Franklin, E. T.; Xia, Y., Uncovering Structural Diversity of Unsaturated Fatty Acyls in Cholesteryl Esters Via Photochemical Reaction and Tandem Mass Spectrometry. J. Am. Soc. Mass. Spectrom. 2017, 28, 1432-1441. (15) Zhang, W.; Chiang, S.; Li, Z.; Chen, Q.; Xia, Y.; Ouyang, Z., Polymer Coating Transfer Enrichment for Direct Mass Spectrometry Analysis of Lipids in Biofluid Samples. Angew. Chem. Int. Ed. 2019, 10.1002/anie.201900011 and 10.1002/ange.201900011. (16) Stinson, C. A.; Xia, Y., A Method of Coupling the Paternò– Büchi Reaction with Direct Infusion ESI-MS/MS for Locating the C=C Bond in Glycerophospholipids. The Analyst 2016, 141, 3696-3704. (17) Tang, F.; Guo, C.; Ma, X.; Zhang, J.; Su, Y.; Tian, R.; Shi, R.; Xia, Y.; Wang, X.; Ouyang, Z., Rapid in Situ Profiling of Lipid C=C Location Isomers in Tissue Using Ambient Mass Spectrometry with Photochemical Reactions. Anal. Chem. 2018, 90, 5612-5619. (18) Deng, J.; Yang, Y.; Xu, M.; Wang, X.; Lin, L.; Yao, Z. P.; Luan, T., Surface-Coated Probe Nanoelectrospray Ionization Mass Spectrometry for Analysis of Target Compounds in Individual Small Organisms. Anal. Chem. 2015, 87, 9923-9930. (19) Deng, J.; Li, W.; Yang, Q.; Liu, Y.; Fang, L.; Guo, Y.; Guo, P.; Lin, L.; Yang, Y.; Luan, T., Biocompatible Surface-Coated Probe for In Vivo, In Situ, and Microscale Lipidomics of Small Biological Organisms and Cells Using Mass Spectrometry. Anal. Chem. 2018, 90, 6936-6944. (20) Jiang, X.; Wang, J.; Guan, Q.; Hu, J.; Xu, J.; Chen, H., Identification of C=C Location of Unsaturated

of lipid C=C locations and isomers for unsaturated lipids in complex biological samples, 2) desirable PB reaction efficiency as well as high electrospray ionization efficiency in new benzophenone PB solvent system, which is suitable for analysis of most lipid species in real biological samples, and 3) determination of lipid C=C locations and isomers for TAGs, which is difficult to achieve using previous reported acetone/water PB reaction system. By using our proposed SCP-PB-nanoESI-MS method, in situ and microscale lipidomics investigation as well as determination of lipid C=C locations and isomers for lipid droplet of Perilla seed and human intestinal tissue were successfully achieved, and in vivo analysis of lipid species and C=C locations for zebrafish was accomplished. The desirable capabilities of our proposed SCPPB-nanoESI-MS method makes it a promising tool for lipidomics investigation.

ASSOCIATED CONTENT Supporting Information Additional information as noted in text. This material is available free of charge via the Internet at http://pubs.acs.org. SCP-PB-nanoESI-MS analysis and Quantitation of C=C location isomers for PCs and TAGs, picture for sampling of lipid droplet from Perilla seed, CID spectra of PB products from lipid droplet of Perilla seed, ESI-MS analysis of lipids from Perilla seed, list of the identified species from lipid droplet of Perilla seed, human intestinal tissue and zebrafish (PDF).

AUTHOR INFORMATION Corresponding Author * E-mail: [email protected]. Tel.: +86-20-37656885-823. * E-mail: [email protected]. Tel.: +86-20-84112958. Notes The authors declare no competing financial interest.

ACKNOWLEDGMENT This research was financially supported by the National Key Research and Development Program of China (2018YFD0900604, 2018YFD0900803), the National Natural Science Foundation of China (No. 21707171, 21625703), Natural Science Foundation of Guangdong Province, China (No. 2017A030310233), Science and Technology Planning Project of Guangzhou City (No. 201804010298), Fundamental Research Funds for the Central Universities (No.171gpy95), and GDAS’ Special Project of Science and Technology Development (2019GDASYL-0302004 and No. 2017GDASCX-0104). We thank Dr. Ruihua Xu and Dr. Zhaolei Zeng in Department of Medical Oncology, Sun Yatsen University Cancer Center, for providing the human intestinal tissue sample.

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Photocycloaddition (Paternò-Büchi Reaction) of Benzophenone to Chiral Allylic Alcohols. J. Am. Chem. Soc. 2000, 122, 2958-2959. (23) Johnson, D. W.; Beckman, K.; Fellenberg, A. J.; Robinson, B. S.; Poulos, A., Monoenoic Fatty Acids in Human Brain Lipids: Isomer Identification and Distribution. Lipids 1992, 27, 177-180. (24) Precht, D.; Molkentin, J., C18:1, C18:2 and C18:3 Trans and Cis Fatty Acid Isomers Including Conjugated Cis Δ9,Trans Δ11 Linoleic Acid (Cla) as well as Total Fat Composition of German Human Milk Lipids. Nahrung 1999, 43, 233-244.

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Figure 4. SCP-PB-nanoESI-MS spectra of a) lipid profile and b) PB product ions of dominant unsaturated lipids from human intestinal tissue. CID spectra of c) [TAG52:2+BP+Na]+ at m/z 1063.8325, and d) [TAG54:3+BP+Na]+ at m/z 1089.8431. 505x718mm (120 x 120 DPI)


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Coupling PaternÃ²-BÃ¼chi Reaction with Surface-Coated Probe

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