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Characterization of pollen by MALDI-TOF lipid profiling
International Journal of Mass Spectrometry 334 (2013) 13–18
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International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Characterization of pollen by MALDI-TOF lipid profiling Miao Liang, Peng Zhang, Xi Shu, Changgeng Liu, Jinian Shu ∗ Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
a r t i c l e
i n f o
Article history: Received 16 April 2012 Received in revised form 20 September 2012 Accepted 24 September 2012 Available online 2 October 2012 Keywords: Pollen Online-MALDI Lipids Bioaerosol
a b s t r a c t In this study, MALDI-TOF lipid profiling was used as an experimental attempt to characterize pollen grains. Magnolia denudata, Lilium brownii var. viridulum, Pinus tabulaeformis, and Populus tomentosa pollen grains were collected as samples. The lipids in pollen grains were extracted by the methods of microwave-assisted formic acid digestion, microwave-assisted 2-aminoethanol solvolysis, and ultrasonic wave-assisted 2-aminoethanol solvolysis. The extracts were analyzed with an online MALDI-TOF mass spectrometer. Membrane-associated phospholipids (phosphatidylserine and phosphatidylcholine) and diacylglycerol in pollen coats were observed. The method of microwave-assisted formic acid digestion presented better lipid profiles. The characteristic mass peaks observed in the experiment may be used as potential signatures for characterizing pollen grains. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Bioaerosols are suspended sediments including allergens and microbial products which contribute to poor air quality as well as infectious diseases and allergies [1–3]. Pollen, as a major component of bioaerosols and a source of allergens, has been a worldwide threat to human health with a rising prevalence suggested by recent epidemiologic data [4–6]. Thus, the need to develop fast and sensitive detection method for airborne pollen grains is constantly growing. Microscopy is the traditional method to identify pollen grains based on morphology, but as such procedures are extremely timeconsuming. Recently, the technologies of the infrared Fourier transform spectroscopy [7], Raman spectroscopy [8] and elastically scattered light imaging [9] have been used as tools for online or rapid pollen identification. Lipids are major components of organisms which play an essential role in structure, energy, and regulation [10–13]. There are several lipidic structures contained in pollen grains, which play key roles and account for a large proportion in pollen exine, pollen coat, intracellular oil bodies and membrane [13,14]. Lipid profiling, the preserved structural skeletons of biological molecules, can be used to study biochemical processes and identify organisms [15–17]. Advances in mass spectrometry instrumentation and technology have greatly increased the characterization of lipid species in organisms [18]. Shotgun lipidomics [19,20] and GC/MS [21,22],
two predominant methods for lipid identification and quantitation, have been used for identifying major lipid components in microorganism samples. Besides, ESI-MS and MALDI have also been applied to analyze the lipids and phospholipids (PLs) extracts or lipids in whole cell bacteria [23–25]. Currently, a large number of methods are available for extracting lipids from plant materials, most of which use organic solvents as the extractants [26,27]. These techniques are usually used for large scale extraction, which are time-consuming and expensive. The need for easier and quicker analyses has prompted the development of new techniques. Among them, microwave-assisted extraction [28] and sonication extraction [29] are the potential alternatives to conventional extraction methods, both of which reduce the volume of extraction solvent required and shorten the sample preparation time as compared to conventional extractions. In this study, the lipids in pollen grains of four species Magnolia denudate (Magnolia), Lilium brownii var. viridulum (Lily), Pinus tabulaeformis (Pine), and Populus tomentosa (Poplar) were extracted with three methods and the lipid extracts were analyzed with a laboratory-built online MALDI-TOF spectrometer. These pollens are the common allergens for the respiratory allergy in Northern China. The aim of this study is to develop an alternative method to characterize pollen grains.
2. Experimental 2.1. Chemicals and pollen grains
∗ Corresponding author. Tel.: +86 010 6284 9508; fax: +86 010 6292 3563. E-mail address: [email protected] (J. Shu). 1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijms.2012.09.007
All solvents (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. and used without further purification.
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M. Liang et al. / International Journal of Mass Spectrometry 334 (2013) 13–18
Fig. 1. Workflow of lipid analysis by online MALDI mass spectrometry.
2,4-Dihydroxybenoic acid (2,4-DHB), 9-aminoacridine (9-AA) and 2-aminoethanol were purchased from Sigma–Aldrich Co., Ltd. The four different pollen grains (Magnolia, Lily, Pine, and Poplar) were collected from the freshly blossoming flowers of mature plant respectively from March to May 2011. Diameters of the pollen grains are as follow: ∼60 m (Magnolia), 50–100 m (Lily), ∼60 m (Pine), and 24–43 m (Poplar). Pollen samples were either used immediately or stored at ∼4 ◦ C for future use. 2.2. Lipid extraction Microwave-assisted formic acid digestion (Method 1): 5 mg of pollen grains suspended in the mixture of formic acid/isopropyl alcohol (2.5 mL/2.5 mL) was microwave-radiated for 5 min with a microwave system (CEM Discover SP). The output power and the holding temperature of the microwave were set at 50 W and 120 ◦ C, respectively. Fig. 1 shows the overall workflow of the experiment. Microwave-assisted 2-aminoethanol solvolysis (Method 2): 5 mg of pollen grains suspended in the mixture of 2aminoethanol/acetonitrile/isopropyl alcohol (1 mL/1 mL/1 mL) was microwave-radiated for 5 min. The output power and the holding temperature of the microwave were set at 50 W and 90 ◦ C, respectively. Ultrasonic wave-assisted 2-aminoethanol solvolysis (Method 3): 10 mg pollen grains suspended in 1 mL of 2-aminoethanol underwent ultrasonication for 10 min to dissolve pollen walls, and then the mixture was extracted with acetonitrile/isopropyl alcohol (2 mL/2 mL) under ultrasonication for another 10 min. The larger sample size used in Method 3 is because the extracting efficiency of Method 3 is lower than those of Methods 1 and 2. The solid materials in the mixtures produced from the above procedures were removed by centrifugation at 6000 rpm for 3 min. An aliquot (5 mL) of the extract was added with 15 mL of 10 mmol/L 2,4-DHB in water/isopropyl alcohol (7.5 mL/7.5 mL) for positive ion MALDI analysis. The same aliquot of lily lipids extracted using Method 3 was also mixed with 9-AA (10 mg/mL in methanol/acetonitrile (60:40, v/v)) for negative ion MALDI analysis.
sample with an incident angle of 50◦ . A quartz lens with f = 750 mm was used to softly focus the laser. The laser spot was approximately 0.5 mm in diameter. The laser pulse energy was about 0.1 mJ/pulse. The ions generated by the laser were detected with a reflectron mass spectrometer characterized by a field free flight distance of 1.2 m, an ion mirror, and a chevron multichannel plate detector. Each mass spectrum was acquired by averaging the ions generated from 128 laser shots. The mass spectra were calibrated by using the ions of ␣-cyclodextrin, -cyclodextrin, and polyethylene glycol (PEG) 1000 cationized by Na+ in the positive ion mode and potassium iodide clusters anionized by I− in the negative ion mode. The calibration error of the mass spectra is within 0.3 Da. 3. Results and discussion Fig. 2 shows the MALDI-TOF mass spectra of pollen lipids extracted from the four pollen samples with Method 1. The ion
2.3. MALDI-TOF MS analysis The analyte/matrix-contained solution obtained above was pneumatically nebulized into suspended droplets, which were instantly analyzed with an online MALDI-TOF mass spectrometer. The liquid consumption of mobilization was ∼0.2 mL/min. The description about the laboratory-built MALDI-TOF mass spectrometer has been introduced in detail elsewhere [30]. The suspended droplets were introduced into the instrument with a nozzle of 0.12 mm orifice combined with an aerodynamic lens assembly tilted at 40◦ from the horizontal plane. The droplets were deposited continuously on a tungsten target plate which also served as the ion repeller of the reflectron mass spectrometer. The deposition spot was ∼0.5 mm in diameter. A 10 Hz pulsed 266 nm Nd:YAG laser (New Wave Research, Polaris III) was used to desorb and ionize the
Fig. 2. MALDI-TOF mass spectra of lipids extracted from pollen grains of Magnolia (A), Lily (B), Pine (C), and Poplar (D) using Method 1. The y axis represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
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Table 1 Tentative assignments and signal intensities (mV) for the MALDI-TOF mass spectra of lipids extracted using Methods 1, 2, and 3. Mag, Lil, Pin, and Pop stand for Magnolia, Lily, Pine, and Poplar, respectively. m/z
Assignment
Method 1 (mV) Mag
603.4 611.5 615.5 631.5 633.5 639.5 647.5 649.5 653.5 655.5 663.4 664.5 671.5 677.5 678.5 689.3 691.5 705.5 726.5 733.6 737.5 754.4 756.6 758.5 774.5 776.5 778.5 780.5 782.5 794.5 796.5 816.5 818.5 820.5 822.5 849.5 851.5 863.5 867.5 869.5 871.5 889.5 891.5 893.5 895.5 911.5 913.5 915.5 917.5 919.5 921.5
[DG(32:0)+K]+ [DG(32:3)+K]+ [DG(33:2)+K]+ [DG(34:2)+K]+ [DG(34:1)+K]+ [DG(35:5)+K]+ [DG(35:1)+K]+ [DG(35:0)+K]+ [DG(36:5)+K]+ [DG(36:4)+K]+ [DG(37:7)+K]+ [DG(36:2)+K]+ [DG(37:3)+K]+ [DG(37:0)+K]+ [DG(38:8)+K]+ [DG(39:8)+K]+ [DG(39:7)+K]+ [DG(40:7)+K]+ [PC(29:2)+K]+ [DG(41:0)+K]+ [PC(30:4)+K]+ [PC(31:2)+K]+ [PC(31:1)+K]+ [PC(31:0)+K]+ [PE(36:6)+K]+ [PC(33:5)+K]+ [PC(33:4)+K]+ [PC(33:3)+K]+ [PC(33:2)+K]+ [PC(34:3)+K]+ [PC(34:2)+K]+ [PC(36:6)+K]+ [PC(36:5)+K]+ [PC(36:4)+K]+ [PC(36:3)+K]+ [PS(38:5)+H+K]+ [PS(38:4)+H+K]+ [PS(39:5)+H+K]+ [PS(39:3)+H+K]+ [PS(39:2)+H+K]+ [PS(39:1)+H+K]+ [PS(39:3)+Na+K]+ [PS(39:2)+Na+K]+ [PS(39:1)+Na+K]+ [PS(39:0)+Na+K]+ [PS(39:2)+2Na+K−H]+ [PS(39:1)+2Na+K−H]+ [PS(39:0)+2Na+K−H]+ [PS(40:6)+2Na+K−H]+ [PS(40:5)+2Na+K−H]+ [PS(40:4)+2Na+K−H]+
Lil
Method 2 (mV) Pin
Pop
Mag
Lil
Method 3 (mV) Pin
Pop
Mag
Lil
Pin
Pop
196.9 105.4 338.8
243.8
26.0
8.7
43.4 17.7
78.8
11.7
48.4
10.6
119.4
37.9
48.2 32.8 16.5 60.6
15.2
32.4 177.1 48.1
66.7 111.3
108.8
417.3
53.7 61.5
44.2 4.7 41.6
87.4 52.3
113.5
134.3
11.8 55.3
28.0
57.0
25.5 62.7 22.4
83.6
6.7 59.6
16.3 42.7
173.9 207.0
7.5 46.6 8.3
39.8 81.7 53.9 50.6 80.5
30.8
5.6 13.8
59.1 45.4 86.6 31.5
21.1 112.2
27.0 32.4 7.2 16.7 13.2 11.7
226.8
7.3
151.1 20.4
17.9 8.6
56.7
6.1 12.8 18.1 4.6
33.3 31.9
177.6 112.3
117.8 115.2 203.1
59.7 142.3 115.7 18.9 35.9 65.1 36.0 19.3
49.3 93.9
15.6 10.9 18.2 23.2 9.8
11.9 30.9
33.2 30.6
111.5
5.5
11.6 13.3
48.6 69.3
23.0 6.9
56.3 55.8
266.2 8.5 20.9 17.9 29.6 75.3
signals below m/z ∼ 600 have been cut off by using an ion gate to avoid the huge matrix ions. Although the four mass spectra shown in Fig. 2 shared some common mass peaks, their specific profiles can be easily distinguished. The assignments of the four mass spectra are listed in Table 1. The most dominant mass peaks were assigned to the following molecular species: DG (32:0), DG (34:2), PS (39:2), and PS (39:1) in Magnolia pollen; PS (39:3), PS (39:2), and PS (40:0) in Lily pollen; DG (34:2), PC (34:2), PS (39:2), PS (39:1), PS (40:6), and PS (40:5) in Pine pollen; DG (37:0), DG (40:7), DG (41:0), PS (39:2), and PS (39:1) in Poplar pollen. The first domination parts (PS) of mass spectra are contributed from intracellular pollen lipids, which is mainly composed of storage triacylglycerol (TAG (39%)) and membrane-associated phospholipids (40%) [31]. Phosphatidylserine (PS) is the major constituent of cell membranes. The form of ions [M+H+K]+ , [M+Na+K]+ and [M+2Na+K−H]+ were observed for PS, which may result from the acidic character of PS [32,33]. Another major constituent of cell membranes,
22.9 56.3 59.6 24.7
109.5 114.8
349.7 85.9 92.4 149.2 89.6 62.7
phosphatidylcholine (PC), yielded the mass peaks at the lower m/z ratio (730–830) than PS. Almost all phospholipids obtained from MALDI mass are highly polyunsaturated because unsaturated fatty acids are necessary for pollen germination. Except for the domination lipids from intracellular pollen, the second domination part diacylglycerol (DG) were mostly in assignment with pollen coat lipids. DG takes up 38.2% in pollen with pollen coat [34]. However, DG was not observed in the mass spectra of Lily pollen lipids extraction. Fig. 3 shows the MALDI-TOF mass spectra of pollen lipids extracted from the four pollen samples using Method 2. The insets shown in Plots A and D of Fig. 3 were obtained by cutting the ions below m/z = 750 with an ion gate and increasing the sensitivity of the ion detector (the voltage of micro-channel plates). DG (34:2), DG (36:5), DG (37:0), DG (40:7), PC (31:0), PC (33:3), PC (34:2), PS (39:3), PS (39:2), PS (39:1), PS (39:0), and PS (40:6) were observed in the extracts of both Methods 1 and 2. The new
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Fig. 3. MALDI-TOF mass spectra of lipids extracted from pollen grains of Magnolia (A), Lily (B), Pine (C), and Poplar (D) using Method 2. The y axis of the figure represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
mass peaks in Fig. 3 were tentatively assigned to DG (32:3), DG (34:1), DG (35:5), DG (35:1), DG (35:0), DG (36:4), DG (36:2), DG (37:3), DG (39:8), PC (33:2), PS (38:5), PS (38:4), PS (39:5), and PE (36:6) (listed in Table 1). Compared with Fig. 2, most mass peaks of PS shown in Fig. 3 were consistent with those shown in Fig. 2, however, their signal intensities were much weaker. More diacylglycerol were observed in Fig. 3, which are pollen coat-associated lipids. The apparent difference between Figs. 2 and 3 may result from the extraction methods. Method 2 was to extract lipids in pollen grains with microwave-assisted 2-aminoethanol solvolysis. 2-Aminoethanol was used for dissolving pollen wall, which is one of the most effective solvents for selectively removing the outer endexine of angiosperm pollen walls without destroying the inner endexine layer [35]. In addition, isopropanol reported as a polar solvent in these methods could extract a variable proportion of the intracellular pollen lipids [36]. For the above reasons, Method 2 may be good for extracting the lipids in pollen coat rather than intracellular pollen. Fig. 4 shows the MALDI-TOF mass spectra of pollen lipids extracted from the four pollen samples using Method 3. Compared with Figs. 2 and 3, the mass peaks of PS (39:2), PS (39:1) were almost absent in Fig. 4 except Lily. Mass spectra of lipids extracted with Methods 2 and 3 shared some common peaks: DG (34:1), DG (35:1), DG (35:0), DG (36:2), PC (31:0), PC (33:3), PC (33:2), PC (34:2), PS (39:3), PS (39:2), PS (39:1), and PS (40:6). Meanwhile, mass spectra of lipids extracted with Methods 1 and 3 shared some common peaks: DG (41:0), PC (31:0), PC (33:4), PC (33:3), PC (34:3), PC (34:2), PC (36:4), PS (39:3), PS (39:2), PS (39:1), and PS (40:6). Besides, more mass peaks ranging from m/z 700 to m/z 850 were observed in Fig. 4. These new mass peaks were assigned
Fig. 4. MALDI-TOF mass spectra of lipids extracted from pollen grains of Magnolia (A), Lily (B), Pine (C), and Poplar (D) using Method 3. The y axis represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
to PC (29:2) and PC (31:1) (listed in Table 1). PC is one major component in tapetosome membranes from which pollen coat is derived [14]. Therefore, Method 3 was good for extracting pollen coat-associated lipids including PC and DG rather than intracellular pollen. The experimental results showed that the extraction method of microwave-assisted formic acid digestion (Method 1) presented better lipid profiles. It should be noted that the assignments are tentative, as the assignments are only based on the ratios of mass to charge (m/z) of the mass peaks. The different lipids which have same molecular weights may overlap each other in the obtained mass spectra. It has been proven that the lipid in organic cells have a large tendency to be adducted with the alkali metal salts, which is a constituent in pollen cells as the form of organic salts. The forms of ions [M+K]+ , [M+H+K]+ , [M+Na+K]+ , and [M+2Na+K−H]+ were observed in the study. In order to assist the assignment of the mass spectra obtained, MALDI analyses on the extracts doped with sodium chloride were conducted. Fig. 5A and B shows the mass spectra of Lily PS from the raw extract and that doped with 2 mmol/L of NaCl, respectively. The raw extracts were obtained with Method 2. New mass peaks at m/z 853.5 and 877.5 in Fig. 5B had a shift of 16 Da to the mass peaks at m/z 869.5 and 893.5 shown in Fig. 5A. In addition, the intensity of the mass peak at m/z 917.5 has been increased relatively. Therefore, we can infer that the mass peaks at m/z 869.5 and 893.5 were contributed from the potassium adducts [M+H+K]+ and [M+Na+K]+ , respectively. While, the mass peak at m/z 917.5 was
M. Liang et al. / International Journal of Mass Spectrometry 334 (2013) 13–18
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solvolysis, and ultrasonic wave-assisted 2-aminoethanol solvolysis. The extracts were analyzed by an online MALDI-TOF mass spectrometer. The membrane-associated phospholipids (PC and PS) and pollen coat lipids (DG) were observed in the experiment. Experimental results show the method of microwave-assisted formic acid digestion presents the mass spectra containing more mass peaks of lipids and higher signal intensity. Compared with the traditional MALDI-MS, this online MALDI-MS avoids the process of analyte/matrix layer preparation and achieves the continuous sampling and detection. The characteristic lipid profiles of pollen may be used as fingerprints to discriminate pollen grains from unidentified chemical powder rapidly. Acknowledgments This research is funded by National High Technology Research and Development Program of China (863 Program, Grant No. 2009AA06Z401) and National Natural Science Foundation of China (Grant No. 20777082). References
Fig. 5. Mass spectra of Lily PS from the raw extract (A) and those doped with 2 mM/L of NaCl (B); Mass spectra of Pine PC from the raw extract (C) and those doped with 2 mM/L of NaCl (D). The y axis represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
contributed from the diadducts of [M+K+2Na−H]+ . The comparisons by this method for other three pollen species presented the same results for PS. The ion form of [M+Na+K]+ was also observed for PC when the extracts are doped with NaCl. Fig. 5C and D shows the mass spectra of Pine PC from the raw extract and that doped with 2 mM/L of NaCl, respectively. The raw extracts were obtained with Method 3. The new mass peaks at m/z 766.5, 780.5, and 804.5 shown in Fig. 5D had a shift of 16 Da to the mass peaks at m/z 782.5, 796.5, and 820.5. We inferred this shift results from adducting of Na instead of K during the ionization process of PC. Therefore, we speculated that most of the mass peaks obtained in the experiment correspond to potassium adducts. The lipids extracted from pollen grains of Lily using Method 3 were also analyzed with online MALDI-TOF MS in the negative ion mode tentatively. 9-Aminoacridine (9-AA) was used as the matrix for the negative ion detection. A strong mass peak at m/z 830.5 was observed and assigned to [PS (39:1)]− . PS (39:1) is the most abundant PS (phosphatidylserine) lipid obtained in spectra of positive mode. This result further verifies the assignment of spectra in the positive mode. 4. Conclusion In this study, lipids of four pollen grains (Magnolia, Lily, Pine and Poplar) were extracted by the methods of microwave-assisted formic acid digestion, microwave-assisted 2-aminoethanol
[1] J. Douwes, P. Thorne, N. Pearce, D. Heederik, Annals of Occupational Hygiene 47 (3) (2003) 187–200. [2] J. Fiegel, R. Clarke, D.A. Edwards, Drug Discovery Today 11 (1–2) (2006) 51–57. [3] L.D. Stetzenbach, Clinical Microbiology Newsletter 20 (19) (1998) 157–161. [4] C. Traidl-Hoffmann, A. Kasche, A. Menzel, T. Jakob, M. Thiel, J. Ring, H. Behrendt, International Archives of Allergy and Immunology 131 (1) (2003) 1–13. [5] G. D’Amato, Clinical and Experimental Allergy 30 (5) (2000) 628–636. [6] G. D’Amato, L. Cecchi, S. Bonini, C. Nunes, I. Annesi-Maesano, H. Behrendt, G. Liccardi, T. Popov, P. van Cauwenberge, Allergy 62 (9) (2007) 976–990. [7] E. Gottardini, S. Rossi, F. Cristofolini, L. Benedetti, Aerobiologia 23 (3) (2007) 211–219. [8] A. Sengupta, M.L. Laucks, E.J. Davis, Applied Spectroscopy 59 (8) (2005) 1016–1023. [9] M. Surbek, C. Esen, G. Schweiger, A. Ostendorf, Journal of Biophotonics 4 (1–2) (2011) 49–56. [10] D.J. Murphy, Progress in Lipid Research 40 (5) (2001) 325–438. [11] I. Coppens, O. Vielemeyer, International Journal for Parasitology 35 (6) (2005) 597–615. [12] A.D. Watson, Journal of Lipid Research 47 (10) (2006) 2101–2111. [13] P. Piffanelli, J.H.E. Ross, D.J. Murphy, Sexual Plant Reproduction 11 (2) (1998) 65–80. [14] I. Hernández-Pinzón, J.H.E. Ross, K.A. Barnes, A.P. Damant, D.J. Murphy, Planta 208 (4) (1999) 588–598. [15] R. Welti, X.M. Wang, Current Opinion in Plant Biology 7 (3) (2004) 337–344. [16] A. Alisi, A. Bartuli, M. Salata, A. Villani, V. Nobili, Clinical Lipidology 6 (4) (2011) 427–436. [17] A. Saliot, J. Laureillard, P. Scribe, M.A. Sicre, Marine Chemistry 36 (1–4) (1991) 233–248. [18] N. Zehethofer, D.M. Pinto, Analytica Chimica Acta 627 (1) (2008) 62–70. [19] C.S. Ejsing, J.L. Sampaio, V. Surendranath, E. Duchoslav, K. Ekroos, R.W. Klemm, K. Simons, A. Shevchenko, Proceedings of the National Academy of Sciences of the United States of America 106 (7) (2009) 2136–2141. [20] L.S. Basconcillo, R. Zaheer, T.M. Finan, B.E. McCarry, Journal of Lipid Research 50 (6) (2009) 1120–1132. [21] A.A. Gharaibeh, K.J. Voorhees, Analytical Chemistry 68 (17) (1996) 2805–2810. [22] L.S. Basconcillo, B.E. McCarry, Journal of Chromatography B 871 (1) (2008) 22–31. [23] B. Fuchs, J. Schiller, European Journal of Lipid Science and Technology 111 (1) (2009) 83–98. [24] Y. Ishida, A.J. Madonna, J.C. Rees, M.A. Meetani, K.J. Voorhees, Rapid Communications in Mass Spectrometry 16 (19) (2002) 1877–1882. [25] P.B.W. Smith, A.P. Snyder, C.S. Harden, Analytical Chemistry 67 (11) (1995) 1824–1830. [26] E.G. Bligh, W.J. Dyer, Canadian Journal of Biochemistry and Physiology 37 (1959) 911–917. [27] J. Folch, M. Lees, G.H.S. Stanley, Journal of Biological Chemistry 226 (1957) 497–509. [28] J.R.J. Pare, G. Matini, J.M.R. Belanger, K. Li, C. Rule, B. Thibert, V. Yaylayan, Z. Liu, D. Mathe, P. Jacquault, Journal of AOAC International 80 (1997) 928–933. [29] A.H. Metherel, A.Y. Taha, H. Izadi, K.D. Stark, Prostaglandins Leukotrienes and Essential Fatty Acids 81 (2009) 417–423. [30] J. Shu, J. Meng, X. Shu, Y. Zhang, S. Gao, B. Yang, J. Gan, Analytical Chemistry 82 (13) (2010) 5906–5909.
18
M. Liang et al. / International Journal of Mass Spectrometry 334 (2013) 13–18
[31] P. Piffanelli, J.H.E. Ross, D.J. Murphy, Plant Journal 11 (3) (1997) 549–562. [32] J. Schiller, J. Arnhold, S. Benard, M. Müller, S. Reichl, K. Arnold, Analytical Biochemistry 267 (1) (1999) 46–56. ´ J. Schiller, M. Müller, S. Benard, S. Reichl, K. Arnold, J. Arnhold, [33] M. Petkovic, Analytical Biochemistry 289 (2) (2001) 202–216.
[34] I. Hernandez-Pinzon, J.H.E. Ross, K.A. Barnes, A.P. Damant, D.J. Murphy, Planta 208 (4) (1999) 588–598. [35] W.J. Kress, Grana 25 (1) (1986) 31–40. [36] G. Bianchi, C. Murelli, E. Ottaviano, Phytochemistry 29 (3) (1990) 739–744.
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International Journal of Mass Spectrometry journal homepage: www.elsevier.com/locate/ijms
Characterization of pollen by MALDI-TOF lipid profiling Miao Liang, Peng Zhang, Xi Shu, Changgeng Liu, Jinian Shu ∗ Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085, China
a r t i c l e
i n f o
Article history: Received 16 April 2012 Received in revised form 20 September 2012 Accepted 24 September 2012 Available online 2 October 2012 Keywords: Pollen Online-MALDI Lipids Bioaerosol
a b s t r a c t In this study, MALDI-TOF lipid profiling was used as an experimental attempt to characterize pollen grains. Magnolia denudata, Lilium brownii var. viridulum, Pinus tabulaeformis, and Populus tomentosa pollen grains were collected as samples. The lipids in pollen grains were extracted by the methods of microwave-assisted formic acid digestion, microwave-assisted 2-aminoethanol solvolysis, and ultrasonic wave-assisted 2-aminoethanol solvolysis. The extracts were analyzed with an online MALDI-TOF mass spectrometer. Membrane-associated phospholipids (phosphatidylserine and phosphatidylcholine) and diacylglycerol in pollen coats were observed. The method of microwave-assisted formic acid digestion presented better lipid profiles. The characteristic mass peaks observed in the experiment may be used as potential signatures for characterizing pollen grains. © 2012 Elsevier B.V. All rights reserved.
1. Introduction Bioaerosols are suspended sediments including allergens and microbial products which contribute to poor air quality as well as infectious diseases and allergies [1–3]. Pollen, as a major component of bioaerosols and a source of allergens, has been a worldwide threat to human health with a rising prevalence suggested by recent epidemiologic data [4–6]. Thus, the need to develop fast and sensitive detection method for airborne pollen grains is constantly growing. Microscopy is the traditional method to identify pollen grains based on morphology, but as such procedures are extremely timeconsuming. Recently, the technologies of the infrared Fourier transform spectroscopy [7], Raman spectroscopy [8] and elastically scattered light imaging [9] have been used as tools for online or rapid pollen identification. Lipids are major components of organisms which play an essential role in structure, energy, and regulation [10–13]. There are several lipidic structures contained in pollen grains, which play key roles and account for a large proportion in pollen exine, pollen coat, intracellular oil bodies and membrane [13,14]. Lipid profiling, the preserved structural skeletons of biological molecules, can be used to study biochemical processes and identify organisms [15–17]. Advances in mass spectrometry instrumentation and technology have greatly increased the characterization of lipid species in organisms [18]. Shotgun lipidomics [19,20] and GC/MS [21,22],
two predominant methods for lipid identification and quantitation, have been used for identifying major lipid components in microorganism samples. Besides, ESI-MS and MALDI have also been applied to analyze the lipids and phospholipids (PLs) extracts or lipids in whole cell bacteria [23–25]. Currently, a large number of methods are available for extracting lipids from plant materials, most of which use organic solvents as the extractants [26,27]. These techniques are usually used for large scale extraction, which are time-consuming and expensive. The need for easier and quicker analyses has prompted the development of new techniques. Among them, microwave-assisted extraction [28] and sonication extraction [29] are the potential alternatives to conventional extraction methods, both of which reduce the volume of extraction solvent required and shorten the sample preparation time as compared to conventional extractions. In this study, the lipids in pollen grains of four species Magnolia denudate (Magnolia), Lilium brownii var. viridulum (Lily), Pinus tabulaeformis (Pine), and Populus tomentosa (Poplar) were extracted with three methods and the lipid extracts were analyzed with a laboratory-built online MALDI-TOF spectrometer. These pollens are the common allergens for the respiratory allergy in Northern China. The aim of this study is to develop an alternative method to characterize pollen grains.
2. Experimental 2.1. Chemicals and pollen grains
∗ Corresponding author. Tel.: +86 010 6284 9508; fax: +86 010 6292 3563. E-mail address: [email protected] (J. Shu). 1387-3806/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijms.2012.09.007
All solvents (analytical grade) were purchased from Sinopharm Chemical Reagent Co., Ltd. and used without further purification.
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Fig. 1. Workflow of lipid analysis by online MALDI mass spectrometry.
2,4-Dihydroxybenoic acid (2,4-DHB), 9-aminoacridine (9-AA) and 2-aminoethanol were purchased from Sigma–Aldrich Co., Ltd. The four different pollen grains (Magnolia, Lily, Pine, and Poplar) were collected from the freshly blossoming flowers of mature plant respectively from March to May 2011. Diameters of the pollen grains are as follow: ∼60 m (Magnolia), 50–100 m (Lily), ∼60 m (Pine), and 24–43 m (Poplar). Pollen samples were either used immediately or stored at ∼4 ◦ C for future use. 2.2. Lipid extraction Microwave-assisted formic acid digestion (Method 1): 5 mg of pollen grains suspended in the mixture of formic acid/isopropyl alcohol (2.5 mL/2.5 mL) was microwave-radiated for 5 min with a microwave system (CEM Discover SP). The output power and the holding temperature of the microwave were set at 50 W and 120 ◦ C, respectively. Fig. 1 shows the overall workflow of the experiment. Microwave-assisted 2-aminoethanol solvolysis (Method 2): 5 mg of pollen grains suspended in the mixture of 2aminoethanol/acetonitrile/isopropyl alcohol (1 mL/1 mL/1 mL) was microwave-radiated for 5 min. The output power and the holding temperature of the microwave were set at 50 W and 90 ◦ C, respectively. Ultrasonic wave-assisted 2-aminoethanol solvolysis (Method 3): 10 mg pollen grains suspended in 1 mL of 2-aminoethanol underwent ultrasonication for 10 min to dissolve pollen walls, and then the mixture was extracted with acetonitrile/isopropyl alcohol (2 mL/2 mL) under ultrasonication for another 10 min. The larger sample size used in Method 3 is because the extracting efficiency of Method 3 is lower than those of Methods 1 and 2. The solid materials in the mixtures produced from the above procedures were removed by centrifugation at 6000 rpm for 3 min. An aliquot (5 mL) of the extract was added with 15 mL of 10 mmol/L 2,4-DHB in water/isopropyl alcohol (7.5 mL/7.5 mL) for positive ion MALDI analysis. The same aliquot of lily lipids extracted using Method 3 was also mixed with 9-AA (10 mg/mL in methanol/acetonitrile (60:40, v/v)) for negative ion MALDI analysis.
sample with an incident angle of 50◦ . A quartz lens with f = 750 mm was used to softly focus the laser. The laser spot was approximately 0.5 mm in diameter. The laser pulse energy was about 0.1 mJ/pulse. The ions generated by the laser were detected with a reflectron mass spectrometer characterized by a field free flight distance of 1.2 m, an ion mirror, and a chevron multichannel plate detector. Each mass spectrum was acquired by averaging the ions generated from 128 laser shots. The mass spectra were calibrated by using the ions of ␣-cyclodextrin, -cyclodextrin, and polyethylene glycol (PEG) 1000 cationized by Na+ in the positive ion mode and potassium iodide clusters anionized by I− in the negative ion mode. The calibration error of the mass spectra is within 0.3 Da. 3. Results and discussion Fig. 2 shows the MALDI-TOF mass spectra of pollen lipids extracted from the four pollen samples with Method 1. The ion
2.3. MALDI-TOF MS analysis The analyte/matrix-contained solution obtained above was pneumatically nebulized into suspended droplets, which were instantly analyzed with an online MALDI-TOF mass spectrometer. The liquid consumption of mobilization was ∼0.2 mL/min. The description about the laboratory-built MALDI-TOF mass spectrometer has been introduced in detail elsewhere [30]. The suspended droplets were introduced into the instrument with a nozzle of 0.12 mm orifice combined with an aerodynamic lens assembly tilted at 40◦ from the horizontal plane. The droplets were deposited continuously on a tungsten target plate which also served as the ion repeller of the reflectron mass spectrometer. The deposition spot was ∼0.5 mm in diameter. A 10 Hz pulsed 266 nm Nd:YAG laser (New Wave Research, Polaris III) was used to desorb and ionize the
Fig. 2. MALDI-TOF mass spectra of lipids extracted from pollen grains of Magnolia (A), Lily (B), Pine (C), and Poplar (D) using Method 1. The y axis represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
M. Liang et al. / International Journal of Mass Spectrometry 334 (2013) 13–18
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Table 1 Tentative assignments and signal intensities (mV) for the MALDI-TOF mass spectra of lipids extracted using Methods 1, 2, and 3. Mag, Lil, Pin, and Pop stand for Magnolia, Lily, Pine, and Poplar, respectively. m/z
Assignment
Method 1 (mV) Mag
603.4 611.5 615.5 631.5 633.5 639.5 647.5 649.5 653.5 655.5 663.4 664.5 671.5 677.5 678.5 689.3 691.5 705.5 726.5 733.6 737.5 754.4 756.6 758.5 774.5 776.5 778.5 780.5 782.5 794.5 796.5 816.5 818.5 820.5 822.5 849.5 851.5 863.5 867.5 869.5 871.5 889.5 891.5 893.5 895.5 911.5 913.5 915.5 917.5 919.5 921.5
[DG(32:0)+K]+ [DG(32:3)+K]+ [DG(33:2)+K]+ [DG(34:2)+K]+ [DG(34:1)+K]+ [DG(35:5)+K]+ [DG(35:1)+K]+ [DG(35:0)+K]+ [DG(36:5)+K]+ [DG(36:4)+K]+ [DG(37:7)+K]+ [DG(36:2)+K]+ [DG(37:3)+K]+ [DG(37:0)+K]+ [DG(38:8)+K]+ [DG(39:8)+K]+ [DG(39:7)+K]+ [DG(40:7)+K]+ [PC(29:2)+K]+ [DG(41:0)+K]+ [PC(30:4)+K]+ [PC(31:2)+K]+ [PC(31:1)+K]+ [PC(31:0)+K]+ [PE(36:6)+K]+ [PC(33:5)+K]+ [PC(33:4)+K]+ [PC(33:3)+K]+ [PC(33:2)+K]+ [PC(34:3)+K]+ [PC(34:2)+K]+ [PC(36:6)+K]+ [PC(36:5)+K]+ [PC(36:4)+K]+ [PC(36:3)+K]+ [PS(38:5)+H+K]+ [PS(38:4)+H+K]+ [PS(39:5)+H+K]+ [PS(39:3)+H+K]+ [PS(39:2)+H+K]+ [PS(39:1)+H+K]+ [PS(39:3)+Na+K]+ [PS(39:2)+Na+K]+ [PS(39:1)+Na+K]+ [PS(39:0)+Na+K]+ [PS(39:2)+2Na+K−H]+ [PS(39:1)+2Na+K−H]+ [PS(39:0)+2Na+K−H]+ [PS(40:6)+2Na+K−H]+ [PS(40:5)+2Na+K−H]+ [PS(40:4)+2Na+K−H]+
Lil
Method 2 (mV) Pin
Pop
Mag
Lil
Method 3 (mV) Pin
Pop
Mag
Lil
Pin
Pop
196.9 105.4 338.8
243.8
26.0
8.7
43.4 17.7
78.8
11.7
48.4
10.6
119.4
37.9
48.2 32.8 16.5 60.6
15.2
32.4 177.1 48.1
66.7 111.3
108.8
417.3
53.7 61.5
44.2 4.7 41.6
87.4 52.3
113.5
134.3
11.8 55.3
28.0
57.0
25.5 62.7 22.4
83.6
6.7 59.6
16.3 42.7
173.9 207.0
7.5 46.6 8.3
39.8 81.7 53.9 50.6 80.5
30.8
5.6 13.8
59.1 45.4 86.6 31.5
21.1 112.2
27.0 32.4 7.2 16.7 13.2 11.7
226.8
7.3
151.1 20.4
17.9 8.6
56.7
6.1 12.8 18.1 4.6
33.3 31.9
177.6 112.3
117.8 115.2 203.1
59.7 142.3 115.7 18.9 35.9 65.1 36.0 19.3
49.3 93.9
15.6 10.9 18.2 23.2 9.8
11.9 30.9
33.2 30.6
111.5
5.5
11.6 13.3
48.6 69.3
23.0 6.9
56.3 55.8
266.2 8.5 20.9 17.9 29.6 75.3
signals below m/z ∼ 600 have been cut off by using an ion gate to avoid the huge matrix ions. Although the four mass spectra shown in Fig. 2 shared some common mass peaks, their specific profiles can be easily distinguished. The assignments of the four mass spectra are listed in Table 1. The most dominant mass peaks were assigned to the following molecular species: DG (32:0), DG (34:2), PS (39:2), and PS (39:1) in Magnolia pollen; PS (39:3), PS (39:2), and PS (40:0) in Lily pollen; DG (34:2), PC (34:2), PS (39:2), PS (39:1), PS (40:6), and PS (40:5) in Pine pollen; DG (37:0), DG (40:7), DG (41:0), PS (39:2), and PS (39:1) in Poplar pollen. The first domination parts (PS) of mass spectra are contributed from intracellular pollen lipids, which is mainly composed of storage triacylglycerol (TAG (39%)) and membrane-associated phospholipids (40%) [31]. Phosphatidylserine (PS) is the major constituent of cell membranes. The form of ions [M+H+K]+ , [M+Na+K]+ and [M+2Na+K−H]+ were observed for PS, which may result from the acidic character of PS [32,33]. Another major constituent of cell membranes,
22.9 56.3 59.6 24.7
109.5 114.8
349.7 85.9 92.4 149.2 89.6 62.7
phosphatidylcholine (PC), yielded the mass peaks at the lower m/z ratio (730–830) than PS. Almost all phospholipids obtained from MALDI mass are highly polyunsaturated because unsaturated fatty acids are necessary for pollen germination. Except for the domination lipids from intracellular pollen, the second domination part diacylglycerol (DG) were mostly in assignment with pollen coat lipids. DG takes up 38.2% in pollen with pollen coat [34]. However, DG was not observed in the mass spectra of Lily pollen lipids extraction. Fig. 3 shows the MALDI-TOF mass spectra of pollen lipids extracted from the four pollen samples using Method 2. The insets shown in Plots A and D of Fig. 3 were obtained by cutting the ions below m/z = 750 with an ion gate and increasing the sensitivity of the ion detector (the voltage of micro-channel plates). DG (34:2), DG (36:5), DG (37:0), DG (40:7), PC (31:0), PC (33:3), PC (34:2), PS (39:3), PS (39:2), PS (39:1), PS (39:0), and PS (40:6) were observed in the extracts of both Methods 1 and 2. The new
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Fig. 3. MALDI-TOF mass spectra of lipids extracted from pollen grains of Magnolia (A), Lily (B), Pine (C), and Poplar (D) using Method 2. The y axis of the figure represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
mass peaks in Fig. 3 were tentatively assigned to DG (32:3), DG (34:1), DG (35:5), DG (35:1), DG (35:0), DG (36:4), DG (36:2), DG (37:3), DG (39:8), PC (33:2), PS (38:5), PS (38:4), PS (39:5), and PE (36:6) (listed in Table 1). Compared with Fig. 2, most mass peaks of PS shown in Fig. 3 were consistent with those shown in Fig. 2, however, their signal intensities were much weaker. More diacylglycerol were observed in Fig. 3, which are pollen coat-associated lipids. The apparent difference between Figs. 2 and 3 may result from the extraction methods. Method 2 was to extract lipids in pollen grains with microwave-assisted 2-aminoethanol solvolysis. 2-Aminoethanol was used for dissolving pollen wall, which is one of the most effective solvents for selectively removing the outer endexine of angiosperm pollen walls without destroying the inner endexine layer [35]. In addition, isopropanol reported as a polar solvent in these methods could extract a variable proportion of the intracellular pollen lipids [36]. For the above reasons, Method 2 may be good for extracting the lipids in pollen coat rather than intracellular pollen. Fig. 4 shows the MALDI-TOF mass spectra of pollen lipids extracted from the four pollen samples using Method 3. Compared with Figs. 2 and 3, the mass peaks of PS (39:2), PS (39:1) were almost absent in Fig. 4 except Lily. Mass spectra of lipids extracted with Methods 2 and 3 shared some common peaks: DG (34:1), DG (35:1), DG (35:0), DG (36:2), PC (31:0), PC (33:3), PC (33:2), PC (34:2), PS (39:3), PS (39:2), PS (39:1), and PS (40:6). Meanwhile, mass spectra of lipids extracted with Methods 1 and 3 shared some common peaks: DG (41:0), PC (31:0), PC (33:4), PC (33:3), PC (34:3), PC (34:2), PC (36:4), PS (39:3), PS (39:2), PS (39:1), and PS (40:6). Besides, more mass peaks ranging from m/z 700 to m/z 850 were observed in Fig. 4. These new mass peaks were assigned
Fig. 4. MALDI-TOF mass spectra of lipids extracted from pollen grains of Magnolia (A), Lily (B), Pine (C), and Poplar (D) using Method 3. The y axis represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
to PC (29:2) and PC (31:1) (listed in Table 1). PC is one major component in tapetosome membranes from which pollen coat is derived [14]. Therefore, Method 3 was good for extracting pollen coat-associated lipids including PC and DG rather than intracellular pollen. The experimental results showed that the extraction method of microwave-assisted formic acid digestion (Method 1) presented better lipid profiles. It should be noted that the assignments are tentative, as the assignments are only based on the ratios of mass to charge (m/z) of the mass peaks. The different lipids which have same molecular weights may overlap each other in the obtained mass spectra. It has been proven that the lipid in organic cells have a large tendency to be adducted with the alkali metal salts, which is a constituent in pollen cells as the form of organic salts. The forms of ions [M+K]+ , [M+H+K]+ , [M+Na+K]+ , and [M+2Na+K−H]+ were observed in the study. In order to assist the assignment of the mass spectra obtained, MALDI analyses on the extracts doped with sodium chloride were conducted. Fig. 5A and B shows the mass spectra of Lily PS from the raw extract and that doped with 2 mmol/L of NaCl, respectively. The raw extracts were obtained with Method 2. New mass peaks at m/z 853.5 and 877.5 in Fig. 5B had a shift of 16 Da to the mass peaks at m/z 869.5 and 893.5 shown in Fig. 5A. In addition, the intensity of the mass peak at m/z 917.5 has been increased relatively. Therefore, we can infer that the mass peaks at m/z 869.5 and 893.5 were contributed from the potassium adducts [M+H+K]+ and [M+Na+K]+ , respectively. While, the mass peak at m/z 917.5 was
M. Liang et al. / International Journal of Mass Spectrometry 334 (2013) 13–18
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solvolysis, and ultrasonic wave-assisted 2-aminoethanol solvolysis. The extracts were analyzed by an online MALDI-TOF mass spectrometer. The membrane-associated phospholipids (PC and PS) and pollen coat lipids (DG) were observed in the experiment. Experimental results show the method of microwave-assisted formic acid digestion presents the mass spectra containing more mass peaks of lipids and higher signal intensity. Compared with the traditional MALDI-MS, this online MALDI-MS avoids the process of analyte/matrix layer preparation and achieves the continuous sampling and detection. The characteristic lipid profiles of pollen may be used as fingerprints to discriminate pollen grains from unidentified chemical powder rapidly. Acknowledgments This research is funded by National High Technology Research and Development Program of China (863 Program, Grant No. 2009AA06Z401) and National Natural Science Foundation of China (Grant No. 20777082). References
Fig. 5. Mass spectra of Lily PS from the raw extract (A) and those doped with 2 mM/L of NaCl (B); Mass spectra of Pine PC from the raw extract (C) and those doped with 2 mM/L of NaCl (D). The y axis represents the relative signal intensity. PS, phosphatidylserine; PC, phosphatidylcholine; DG, diacylglycerol.
contributed from the diadducts of [M+K+2Na−H]+ . The comparisons by this method for other three pollen species presented the same results for PS. The ion form of [M+Na+K]+ was also observed for PC when the extracts are doped with NaCl. Fig. 5C and D shows the mass spectra of Pine PC from the raw extract and that doped with 2 mM/L of NaCl, respectively. The raw extracts were obtained with Method 3. The new mass peaks at m/z 766.5, 780.5, and 804.5 shown in Fig. 5D had a shift of 16 Da to the mass peaks at m/z 782.5, 796.5, and 820.5. We inferred this shift results from adducting of Na instead of K during the ionization process of PC. Therefore, we speculated that most of the mass peaks obtained in the experiment correspond to potassium adducts. The lipids extracted from pollen grains of Lily using Method 3 were also analyzed with online MALDI-TOF MS in the negative ion mode tentatively. 9-Aminoacridine (9-AA) was used as the matrix for the negative ion detection. A strong mass peak at m/z 830.5 was observed and assigned to [PS (39:1)]− . PS (39:1) is the most abundant PS (phosphatidylserine) lipid obtained in spectra of positive mode. This result further verifies the assignment of spectra in the positive mode. 4. Conclusion In this study, lipids of four pollen grains (Magnolia, Lily, Pine and Poplar) were extracted by the methods of microwave-assisted formic acid digestion, microwave-assisted 2-aminoethanol
[1] J. Douwes, P. Thorne, N. Pearce, D. Heederik, Annals of Occupational Hygiene 47 (3) (2003) 187–200. [2] J. Fiegel, R. Clarke, D.A. Edwards, Drug Discovery Today 11 (1–2) (2006) 51–57. [3] L.D. Stetzenbach, Clinical Microbiology Newsletter 20 (19) (1998) 157–161. [4] C. Traidl-Hoffmann, A. Kasche, A. Menzel, T. Jakob, M. Thiel, J. Ring, H. Behrendt, International Archives of Allergy and Immunology 131 (1) (2003) 1–13. [5] G. D’Amato, Clinical and Experimental Allergy 30 (5) (2000) 628–636. [6] G. D’Amato, L. Cecchi, S. Bonini, C. Nunes, I. Annesi-Maesano, H. Behrendt, G. Liccardi, T. Popov, P. van Cauwenberge, Allergy 62 (9) (2007) 976–990. [7] E. Gottardini, S. Rossi, F. Cristofolini, L. Benedetti, Aerobiologia 23 (3) (2007) 211–219. [8] A. Sengupta, M.L. Laucks, E.J. Davis, Applied Spectroscopy 59 (8) (2005) 1016–1023. [9] M. Surbek, C. Esen, G. Schweiger, A. Ostendorf, Journal of Biophotonics 4 (1–2) (2011) 49–56. [10] D.J. Murphy, Progress in Lipid Research 40 (5) (2001) 325–438. [11] I. Coppens, O. Vielemeyer, International Journal for Parasitology 35 (6) (2005) 597–615. [12] A.D. Watson, Journal of Lipid Research 47 (10) (2006) 2101–2111. [13] P. Piffanelli, J.H.E. Ross, D.J. Murphy, Sexual Plant Reproduction 11 (2) (1998) 65–80. [14] I. Hernández-Pinzón, J.H.E. Ross, K.A. Barnes, A.P. Damant, D.J. Murphy, Planta 208 (4) (1999) 588–598. [15] R. Welti, X.M. Wang, Current Opinion in Plant Biology 7 (3) (2004) 337–344. [16] A. Alisi, A. Bartuli, M. Salata, A. Villani, V. Nobili, Clinical Lipidology 6 (4) (2011) 427–436. [17] A. Saliot, J. Laureillard, P. Scribe, M.A. Sicre, Marine Chemistry 36 (1–4) (1991) 233–248. [18] N. Zehethofer, D.M. Pinto, Analytica Chimica Acta 627 (1) (2008) 62–70. [19] C.S. Ejsing, J.L. Sampaio, V. Surendranath, E. Duchoslav, K. Ekroos, R.W. Klemm, K. Simons, A. Shevchenko, Proceedings of the National Academy of Sciences of the United States of America 106 (7) (2009) 2136–2141. [20] L.S. Basconcillo, R. Zaheer, T.M. Finan, B.E. McCarry, Journal of Lipid Research 50 (6) (2009) 1120–1132. [21] A.A. Gharaibeh, K.J. Voorhees, Analytical Chemistry 68 (17) (1996) 2805–2810. [22] L.S. Basconcillo, B.E. McCarry, Journal of Chromatography B 871 (1) (2008) 22–31. [23] B. Fuchs, J. Schiller, European Journal of Lipid Science and Technology 111 (1) (2009) 83–98. [24] Y. Ishida, A.J. Madonna, J.C. Rees, M.A. Meetani, K.J. Voorhees, Rapid Communications in Mass Spectrometry 16 (19) (2002) 1877–1882. [25] P.B.W. Smith, A.P. Snyder, C.S. Harden, Analytical Chemistry 67 (11) (1995) 1824–1830. [26] E.G. Bligh, W.J. Dyer, Canadian Journal of Biochemistry and Physiology 37 (1959) 911–917. [27] J. Folch, M. Lees, G.H.S. Stanley, Journal of Biological Chemistry 226 (1957) 497–509. [28] J.R.J. Pare, G. Matini, J.M.R. Belanger, K. Li, C. Rule, B. Thibert, V. Yaylayan, Z. Liu, D. Mathe, P. Jacquault, Journal of AOAC International 80 (1997) 928–933. [29] A.H. Metherel, A.Y. Taha, H. Izadi, K.D. Stark, Prostaglandins Leukotrienes and Essential Fatty Acids 81 (2009) 417–423. [30] J. Shu, J. Meng, X. Shu, Y. Zhang, S. Gao, B. Yang, J. Gan, Analytical Chemistry 82 (13) (2010) 5906–5909.
18
M. Liang et al. / International Journal of Mass Spectrometry 334 (2013) 13–18
[31] P. Piffanelli, J.H.E. Ross, D.J. Murphy, Plant Journal 11 (3) (1997) 549–562. [32] J. Schiller, J. Arnhold, S. Benard, M. Müller, S. Reichl, K. Arnold, Analytical Biochemistry 267 (1) (1999) 46–56. ´ J. Schiller, M. Müller, S. Benard, S. Reichl, K. Arnold, J. Arnhold, [33] M. Petkovic, Analytical Biochemistry 289 (2) (2001) 202–216.
[34] I. Hernandez-Pinzon, J.H.E. Ross, K.A. Barnes, A.P. Damant, D.J. Murphy, Planta 208 (4) (1999) 588–598. [35] W.J. Kress, Grana 25 (1) (1986) 31–40. [36] G. Bianchi, C. Murelli, E. Ottaviano, Phytochemistry 29 (3) (1990) 739–744.