Anal. Chem. 2003, 75, 4514-4518
Direct Analysis of Lipids in Single Zooplankter Individuals by Matrix-Assisted Laser Desorption/ Ionization Mass Spectrometry Yasuyuki Ishida,† Oh Nakanishi,‡ Shingo Hirao,‡ Shin Tsuge,‡ Jotaro Urabe,§ Tatsuki Sekino,| Masami Nakanishi,| Takashi Kimoto,⊥ and Hajime Ohtani*,‡
Research Center for Advanced Energy Conversion, and Department of Applied Chemistry, Graduate School of Engineering, Nagoya University, Nagoya 464-8603, Japan, Center for Ecological Research, Kyoto University, Otsu, 520-2113, Japan, Research Institute of Humanity and Nature, Kyoto 602-0878, Japan, and Research Institute of Oceano-Chemistry, Osaka 543-0024, Japan
A highly sensitive method to analyze the intact lipids in a single zooplankter individual at the level of a few tenths of a microgram was developed by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) combined with a direct sampling technique. The sampling procedure involved (1) putting a zooplankter individual sample onto the MALDI sample plate, (2) cutting the sample into a few pieces by means of tweezers, (3) depositing aliquots of matrix and cationization reagent solutions on the zooplankter sample, and (4) irradiating with a N2 laser to cause MALDI. By using this technique, the mass spectra of the single zooplankter samples showed a series of ions generated from phospholipids with 34 or 36 carbons in the acyl groups and neutral lipids such as triglycerides and diacylglyceryl ethers with 5054 carbons in their acyl and alkenyl groups. Accordingly, this method enabled us to estimate the relative quantity between “structured lipids” (phospholipids) and “storage lipids” (neutral lipids) in an individual zooplankter, which should give us a good clue to elucidate the roles of each class of lipids in its growth. The amount and type of lipid contained within zooplankton tissues are often a good reflection of their recent feeding success and selectivity.1-6 Various other external factors such as water temperature7 and internal factors such as developmental stage7 were also reported to influence zooplankton lipid content. In the * To whom correspondence should be addressed: (phone) +81-52-789-3560; (fax) +81-52-789-4666; (e-mail)
[email protected]. † Research Center for Advanced Energy Conversion, Nagoya University. ‡ Department of Applied Chemistry, Nagoya University. § Kyoto University. | Research Institute of Humanity and Nature. ⊥ Research Institute of Oceano-Chemistry. (1) Arts, M. T. In Lipids in Freshwater Ecosystems; Arts, M. T., Wainman, B. C., Eds.; Springer-Verlag: New York, 1999; Chapter 4. (2) Lee, R. F.; Nevenzel, J. C.; Paffenhofer, G.-A. Mar. Biol. 1971, 9, 99-108. (3) Falk-Peterson, S.; Sargent, J. R.; Tande, K. S. Polar Biol. 1987, 8, 115120. (4) Bourdier, G. G.; Amblard, C. A. J. Plankton Res. 1989, 11, 1201-1212. (5) Demott, W. R.; Muller-Navarra, D. C. Freshwater Biol. 1997, 38, 649-664. (6) Weers, P. M. M.; Siewertsen, K.; Gulati, R. D. Freshwater Biol. 1997, 38, 731-738. (7) Farkas, T. Comp. Biochem. Physiol. 1979, 64B, 71-76.
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field of environmental and biological sciences, therefore, the analysis of lipid components in zooplankton often provides a good clue to evaluate (1) the lipid metabolism occurring in vivo, (2) the food web and cycling of carbon in hydrosphere, and (3) the quality of the environment. In particular, it has been of interest to analyze the lipid components contained in an individual zooplankter in order to evaluate their role in the growth of the zooplankter in detail. Recently, the authors have successfully applied thermally assisted hydrolysis and methylation-gas chromatography (THMGC) in the presence of organic alkali to the lipid analysis for an individual zooplankter of a few tenths of a microgram.9-12 In this technique, the total amount of fatty acid components and their composition in an individual zooplankter were determined on the basis of the peak intensities of fatty acid methyl esters observed on the chromatogram. Although this method provided good information for discriminative analysis among individual zooplankters without applying any sample pretreatment, it was impossible to analyze the intact structures of the lipid molecules in the zooplankters because of inevitable hydrolysis of ester linkages in the lipids. On the other hand, matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) has been widely utilized as a soft ionization MS technique, which often provides mass spectra mainly composed of molecular-related ion peaks with little or no fragmentation for various biopolymers and synthetic macromolecules. MALDI-MS has also gained popularity for the rapid analysis of lipids in various biological samples such as plant seeds,13 human neutrophils,14 human plasma,15 and bacteria.16 However, these approaches generally involved a solvent extraction (8) Kattner, G.; Krause, M. Mar. Biol. 1987, 96, 511-518. (9) Ishida, Y.; Isomura, S.; Tsuge, S.; Ohtani, H.; Sekino, T.; Nakanishi, M.; Kimoto, T. Analyst 1996, 121, 853-856. (10) Sekino, T.; Nakanishi, M.; Ishida, Y.; Isomura, S.; Tsuge, S.; Ohtani, H.; Kimoto, T. Freshwater Biol. 1997, 38, 611-618. (11) Ishida, Y.; Yokoi, H.; Isomura, S.; Ohtani, H.; Tsuge, S.; Sekino, T.; Nakanishi, M.; Kimoto, T. J. Chromatogr., B 1998, 716, 39-45. (12) Nakanishi, O.; Hirao, S.; Ishida, Y.; Tsuge, S.; Ohtani, H.; Urabe, J.; Sekino, T.; Nakanishi, M.; Kimoto, T. J. Anal. Appl. Pyrolysis 2003, 68/69, 187195. (13) Asbury, G. R.; Al-Saad, K.; Siems, W. F.; Hannan, R. M.; Hill, H. H., Jr. J. Am. Soc. Mass Spectrom. 1999, 10, 983-991. (14) Schiller, J.; Arnhold, J.; Benard, S.; Muller, M.; Reichl, S.; Arnold, K. Anal. Biochem. 1999, 267, 46-56. 10.1021/ac030072j CCC: $25.00
© 2003 American Chemical Society Published on Web 07/30/2003
procedure of the lipids from the sample matrix prior to the MALDI-MS measurements. So far, therefore, this technique has not been applied to the lipid characterization of a single zooplankter at the level of a few tenths of a microgram because fairly large amounts (10-15 mg) of the samples are usually requested for the extraction procedure. Nowadays, a sampling procedure for MALDI-MS called “onprobe sample pretreatment” is often utilized for direct detection of intact proteins and phospholipids in whole bacterial cells.17-20 In this technique, live bacterial colonies are transferred directly from agar surfaces to the MALDI sample plate and then overlaid with a microvolume of matrix solution prior to irradiation with a N2 laser. The resulting molecular ions of intact phospholipids and proteins observed on the mass spectra were also used as the key peaks for rapid discrimination among the species of bacteria. In this work, the authors developed a novel method to analyze the intact lipids in an individual zooplankter by means of MALDI-MS combined with a direct sampling procedure such as the “on-probe sample pretreatment”. By this technique, the relative quantity of each class of lipid such as “structured lipids” (phospholipids) and “storage lipids” (neutral lipids) was estimated at the individual level for the first time. EXPERIMENTAL SECTION Materials. Daphnia galeata isolated from Lake Biwa were cultured individually in 50-mL bottles containing COMBO21 medium with 2 mg of C L-1 of green algae, Scenedesmus actus, as food at 20 °C for 1-2 weeks and were used in this work. Growth medium and feeding suspension were changed every day. These individual samples were weighed in the range from 10 to 80 µg of dry weight base and their physical sizes were ∼1 mm. These zooplankter samples were cultured simultaneously under conditions identical with those used in our previous report for THMGC measurements.12 In addition, a finely powdered plankter sample was used to evaluate the precision of the MALDI-MS method. This homogenized sample was prepared from ∼20 individuals (a few milligrams) of Daphnia magna, collected in Lake Biwa, by grinding in a mortar. Tripalmitin (Sigma), 1,2-dipalmitoylphosphatidylcholine (Sigma), 1,2-dioleylphosphatidylglycerol (Sigma), and 1,2-dipalmitoylphosphatidylethanolamine (Sigma) were used as standard lipids to identify the lipid components in plankton. According to the previous reports on MALDI-MS measurements of lipids,14,15,22,23 2,5-dihydroxybenzoic acid (DHB; Aldrich) was used as a matrix reagent. The DHB matrix solution (500 mM) (15) Schiller, J.; Arnhold, J.; Glander, H.-J.; Arnold, K. Chem. Phys. Lipids 2000, 106, 145-156. (16) Ho, Y.-P.; Fenselau, C. Anal. Chem. 1998, 70, 4890-4895. (17) Claydon, M. A.; Davey, S. N.; Edwards-Jones, V.; Gordon, D. B. Nat. Biotechnol. 1996, 14, 1584-1586. (18) Evason, D. J.; Claydon, M. A.; Gordon, D. B. Rapid Commun. Mass Spectrom. 2000, 14, 669-672. (19) Madonna, A. J.; Basile, F.; Ferrer, I.; Meetani, M. A.; Rees, J. C.; Voorhees, K. J. Rapid Commun. Mass Spectrom. 2000, 14, 2220-2229. (20) Ishida, Y.; Madonna, A. J.; Meetani, M. A.; Rees, J. C.; Voorhees, K. J. Rapid Commun. Mass Spectrom. 2002, 16, 1877-1882. (21) Kilham, S. S.; Kreeger, D. A.; Lynn S. G.; Goulden, C. E.; Herrera, L. Hydrobiologia 1998, 377, 147-159. (22) Marto, J. A.; White, F. M.; Seldomridge, S.; Marshall, A. G. Anal. Chem. 1995, 67, 3979-3984. (23) Muller, M.; Schiller, J.; Petkovic, M.; Oehrl, W.; Heinze, R.; Wetzker, R.; Arnold, K.; Arnhold, J. Chem. Phys. Lipids 2001, 110, 151-164.
Figure 1. Procedures for direct MALDI-MS measurements of an individual zooplankter. (1) A single zooplankter sample is spotted onto the MALDI sample plate; (2) the sample is cut into a few pieces carefully by using sharp tweezers under a magnifier; (3) matrix and cationization reagents are sequentially deposited onto the sample spot; (4) the MALDI-MS measurement is performed by irradiating with a N2 laser to the sample.
was prepared by dissolving ∼80 mg of the matrix reagent in 1 mL of the solvent (2:1 mixture of chloroform/MeOH). Sodium iodide (Wako), dissolved in the same solvent at a concentration of 30 mM, was used as a cationization reagent to form selectively the sodium adduct ions [M + Na+] of the analytes. Sampling Procedure. Figure 1 illustrates the procedures for direct lipid analysis in a single zooplankter sample by MALDIMS. First, an individual zooplankter is directly deposited onto the MALDI sample plate (1) and then cut into a few pieces by means of sharp tweezers under a magnifier to assist extraction of the lipid components toward the zooplankter’s surface and deeper penetration of the matrix reagent (2). Next, 1-µL droplets of the matrix and cationization reagent solutions are successively deposited onto the surface of the zooplankter sample (3) prior to irradiation with the N2 laser to cause MALDI (4). MALDI-MS Measurement. The MALDI-MS measurements were performed using a Voyager DE-RP time-of-flight mass spectrometer (Applied Biosystems) equipped with a pulsed nitrogen laser (r ) 337 nm, 3-ns pulse width, and 3-Hz frequency) and a delayed extraction ion source. The laser beam intensity was experimentally attenuated to the level slightly beyond the threshold for lipid ionization. Ions generated by the laser desorption were introduced into the flight tube with an accelerating voltage of 10 kV operated in a positive reflectron mode. The delay time was set at 100 ns. All mass spectra were acquired by averaging 100 individual laser shots. Mass calibration was accomplished using tripalmitin and 1,2-dipalmitoylphosphatidylcholine as external standards. RESULTS AND DISCUSSION Figure 2 shows a typical MALDI mass spectrum of a single D. galeata sample weighing ∼30 µg, observed by the direct sampling procedure described above. In this spectrum, a series of ions corresponding to the masses of phospholipids and neutral lipids were separately observed in the mass range of m/z 740Analytical Chemistry, Vol. 75, No. 17, September 1, 2003
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Figure 2. Typical MALDI-MS spectrum of an individual D. galeata sample. PC, TAG, and DAGE refer phosphatidylcholines, triacylglycerides and diacylglyceryl ether, respectively. Table 1. Assignment of Representative Peaks in the Spectrum of Figure 2 m/z
relative intensitiesb
assignmentc
777a 779a 781a 783a 785a 799 801 803 805 807 809 811
46.6 100 71.9 74.0 63.0 23.3 46.6 39.0 66.4 54.8 32.9 24.7
(a) Phosphatidylcholines PC (C34:4) + Na+ PC (C34:3) + Na+ PC (C34:2) + Na+ PC (C34:1) + Na+ PC (C34:0) + Na+ PC (C36:6) + Na+ PC (C36:5) + Na+ PC (C36:4) + Na+ PC (C36:3) + Na+ PC (C36:2) + Na+ PC (C36:1) + Na+ PC (C36:0) + Na+
860 862 864 866 868 870 872 874 876 878 880 882 884 886
19.8 22.6 24.5 26.6 34.2 45.1 29.8 37.0 44.1 42.4 32.9 27.2 16.2 9.5
(b) Neutral Lipids DAGE (C52:6) + Na+ DAGE (C52:5) + Na+ DAGE (C52:4) + Na+ DAGE (C52:3) + Na+ DAGE (C52:2) + Na+ DAGE (C52:1) + Na+ TAG (C52:7) + Na+ TAG (C52:6) + Na+ TAG (C52:5) + Na+ TAG (C52:4) + Na+ TAG (C52:3) + Na+ TAG (C52:2) + Na+ TAG (C52:1) + Na+ TAG (C52:0) + Na+
possible fatty acid combinationd (16:1, 18:3) (16:0, 18:4) (16:0, 18:3) (16:1, 18,2) (16:0, 18:2) (16:1, 18:1) (16:0, 18:1) (16:1, 18:0) (16:0, 18:0) (18:3, 18:3) (18:2, 18:4) (18:1, 18:4) (16:0, 20:5) (18:1, 18:3) (18:0, 18:4) (18:0, 18:3) (18:1, 18:2) (18:0, 18:2) (18:1, 18:1) (18:0, 18:1) (18:0, 18:0)
(18:2, 18:3) (16:0, 20:5) (18:1, 18:3) (18:0, 18:4) (18:0, 18:3) (18:1, 18:2) (18:0, 18:2) (18:1, 18:1) (18:0, 18:1) (18:0, 18:0) (16:0, 18:3, 18:4) (14:0, 16:1, 22:6) (16:0, 18:3, 18:3) (14:0, 16:0, 22:6) (16:0, 18:1, 18:4) (14:0, 18:0, 20:5) (16:0, 18:1, 18:3) (16:1, 18:0, 18:3) (16:0, 18:0, 18:3) (16:0, 18:1, 18:2) (16:0, 18:1, 18:1) (16:1, 18:0, 18:1) (16:0, 18:0, 18:1) (16:1, 18:0, 18:0) (16:0, 18:0, 18:0)
a Protonated adduct ions of PC (C36) components might be overlapped to some extent. b Normalized by the peak intensitiy of [PC (C34:3) + Na+], which was the most intense one on the spectrum. c PC, TAG, and DAGE refer phosphatidylcholine, triacylglyceride, and diacylglyceryl ether, respectively. d Inferred based on the reported fatty acid distribution for the given D. galeata samples by THM-GC.12 The number 18:3, for example, indicates the carbon number (18) and three double bonds.
820 and 820-920, respectively. The result thus observed indicates that this technique can be used for the highly sensitive and selective detection of intact phospholipids and neutral lipids, referred to as “structured lipids”, the major components of cell membranes, and as “storage lipids”, the energy reservoirs, respectively, directly from an individual zooplankter. 4516 Analytical Chemistry, Vol. 75, No. 17, September 1, 2003
Figure 3 shows expanded mass spectra of the D. galeata sample in the structured lipids region (m/z 760-820) (a) and the neutral lipids region (m/z 820-920) (b). Both of the spectra showed a series of ions generated from lipid components with enough resolution to separate those peaks isotopically. To identify the peaks observed in the structured lipids region in detail, a series
Figure 3. Expanded MALDI-MS spectra of a single D. galeata sample in (a) phospholipid region and (b) neutral lipid region. The peaks marked with circles (O), squares (0), and triangles (4) refer to phosphatidylcholines, triacylglycerides, and diacylglyceryl ether, respectively. Cxx indicates the component containing totally xx carbons in its acyl (+ alkenyl) groups.
of standard phospholipid samples, phosphatidylcholines (PCs), phosphatidylethanolamines (PEs), and phosphatidylglycerols (PGs), known to be the main classes of phospholipids present in zooplankton,24-26 were also subjected to MALDI-MS measurement under the same conditions. In the spectra of these standard samples, the molecular ions were mainly observed in the form of their sodium adducts [M + Na+], along with weaker (in less than (24) Lee, R. F. Mar. Biol. 1974, 26, 313-318. (25) Lee, R. F.; Nevenzel, J. C.; Lewis, A. G. Lipids 1974, 9, 891-898. (26) Sargent, J. R.; Lee, R. F. Mar. Biol. 1975, 31, 15-23.
half-height) ones corresponding to their proton adducts [M + H+]. Based on the masses of the molecular ions for the standard phospholipids, the main compounds of the peaks marked with circles in the mass spectrum (a), e.g., m/z 779, 781, 783, 801, and 805, were estimated to be PCs including total 34 or 36 carbons in the two acyl groups [PC (C34 or 36)]. However, the peaks appearing in the smaller m/z range around m/z 750-760 were presumed to be [PC (C34) + H+] rather than [PC (C32) + Na+] considering the relative yield of the protonated ions observed for the standard lipids. Meanwhile, in the mass spectrum of the neutral lipids region (m/z 820-920) (b), the peaks marked with squares were assumed to be mostly the sodium adduct ions of triacylglycerides (TAGs) consisting of total 50-54 carbons in the three acyl groups [TAG (C50-C54)] based on the MALDI-MS measurements of the standard TAG sample. Moreover, in a given carbon number region for the neutral lipids, some additional peaks marked with triangles were accompanied at the slightly smaller m/z values than those of TAGs as shown in spectrum b. For example, in the C52 region, a series of ions marked with triangles appeared at m/z 866, 868, 870, etc. in addition to those for TAGs (m/z 876, 878, 890, etc.). These components were supposed to be the sodium adduct ions of diacylglyceryl ethers (DAGEs), which were reported to be present as one of the important lipid components in some zooplankton species.27 Here, taking into consideration the fatty acid compositions in the same set of D. galeata samples obtained by THM-GC,12 the observed peaks in the MALDI mass spectrum in Figure 3 were interpreted in detail in terms of the carbon number and the degree of unsaturation of the respective acyl groups. The observed results by THM-GC suggested that the given zooplankter samples contained commonly C14-C22 saturated and unsaturated fatty acid components including highly polyunsaturated ones such as eicosapentaenoic acid (C20:5) and docosahexaenoic acid (C22: 6). Furthermore, it was revealed that fairly large contents of DAGEs were also present in the examined plankter samples mostly in the form of the hexadecenyl (16:1) ether derivatives. Table 1 illustrates the possible assignment of representative peaks in PC (C34), PC (C36), TAG (C52), and DAGE (C52) regions of the mass spectrum in Figure 3 on the assumption that all the components were observed as their sodium adduct ions. In this table, the total carbon numbers and the degree of unsaturation in acyl groups are summarized together with their relative peak intensities and the most probable combinations of fatty acid components. The suggested fatty acid combinations for the lipid components containing high numbers of total double bonds are consistent with the fact that polyunsaturated fatty acid residues such as C20:5 and C22:6 were present to some extent as well as saturated and monounsaturated ones in their acyl groups as was confirmed by THM-GC.12 Finally, various D. galeata individual samples were similarly subjected to the MALDI-MS measurements using the direct sampling procedure to quantify the relative peak intensities between sum of the structured lipids (PCs) region (m/z 750820) and that of the storage lipids (neutral lipids) region (m/z 820-920). Prior to the measurement of individual samples, the reproducibility of the observed contents between two classes of (27) Lee, R. F. J. Fish. Res. Board Can. 1974, 31, 1577-1582.
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Table 2. Relative Peak Intensities between Structured and Storage Lipids Observed in the MALDI Mass Spectra of Five Zooplankter Individuals observed relative peak intensities (%) structured lipids storage lipids zooplankter dry wt samples (µg) PCs TAGs DAGEs (subtotal) A B C D E
83.7 27.6 26.8 21.5 18.9
47.8 49.9 59.6 56.4 60.9
31.5 28.3 23.6 25.8 23.2
20.7 21.8 16.8 17.8 15.9
(52.2) (50.1) (40.4) (43.6) (39.1)
total 100.0 100.0 100.0 100.0 100.0
the lipids obtained by this method was assessed through MALDIMS measurements of the homogenized plankter powder sample weighing ∼100 µg, consistent with the typical dry weight of an individual zooplankter sample. About 15% of the relative standard deviation for 10 repeated runs thus obtained suggests that the observed relative abundances of the lipid components in a single zooplankter can be evaluated in terms of their individual differences. The amounts obtained for five individual samples are summarized in Table 2 together with the dry weights of the samples. Among these individuals, the relative amount of neutral lipids
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generally increases with increase in their dry weights mainly due to the contribution of TAGs. This observation suggests that the larger zooplankters might store the ingested lipid components more preferably in the form of neutral lipids as energy reservoirs for egg production and respiratory movement. As described above, highly sensitive detection of intact lipids in an individual zooplankter was achieved by MALDI-MS combined with the direct sampling procedure. This technique allowed us to observe the structured lipids (phospholipids) and storage lipids (neutral lipids) in single zooplankters separately in the resulting MALDI mass spectra. By applying this method to lipid analysis in individual zooplankters grown under various environmental conditions, the roles of each class of lipid in growth and reproduction could be elucidated. Further work along this line is in progress. ACKNOWLEDGMENT This work was supported in part by the 21st Century COE Program “Nature-Guided Materials Processing” of the Ministry of Education, Culture, Sports, Science and Technology of Japan. Received for review February 24, 2003. Accepted May 22, 2003. AC030072J
