In the Laboratory
Analysis of Phospholipid Mixtures from Biological Tissues by Matrix-Assisted Laser Desorption and Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS): A Laboratory Experiment € rgen Schiller,* Rosmarie Su € ß, and Kristin Teuber Mandy Eibisch, Beate Fuchs, Ju University of Leipzig, Faculty of Medicine, Institute of Medical Physics and Biophysics, Ha.rtelstrasse 16-18, D-04107 Leipzig, Germany *
[email protected] Interest in lipids and particularly phospholipids (PL) is increasing because these molecules represent abundant membrane constituents of cells as well as important second messengers and thus possess significant diagnostic potential (1). Additionally, PL ionize well and are, therefore, often in the focus of studies related to mass spectrometry (MS) imaging studies of biological tissues (2). MS techniques are highly established in the field of lipid analysis owing to the high sensitivity and mass resolution of these techniques (3). As it is desirable to detect the intact lipid molecules with their correct masses (but not any fragment ions derived thereof), soft ionization MS techniques are the methods of choice. The two most important techniques are electrospray ionization (ESI) (4) and matrix-assisted laser desorption and ionization (MALDI) MS (5), often with a time-of-flight mass (TOF) analyzer (6). In comparison to the “classical” ionization MS method, electron ionization (EI) MS, soft ionization MS exhibits an important difference (7): EI normally produces radical ions by abstraction of one electron from the analyte molecule. In contrast, ESI and MALDI generate ions by the addition of one cation, for instance, a proton or an alkali metal ion. This clearly alters the mass in comparison to the neutral molecule and, to indicate this fact, the terms “quasimolecular ions” or “adducts” are often used. The addition of, for example, Hþ to the analyte molecule depends on the acidity of the applied matrix (normally an organic acid) and the environment and on the basicity of the analyte. Although the determination of pK values in the gas phase (where ionization of MALDI MS most probably occurs) is a challenging task (8), it is obvious that in the presence of matrix compounds with different acidities or basicities a certain analyte will give different ion yields. Thus, different analytes will be detectable with different sensitivities. Using some selected, synthetic lipids and some organic extracts from different easily available biological materials (egg yolk and liver extracts), we show that great care is required to interpret the MALDI mass spectra of complex lipid mixtures because only selected PL classes are detectable (9). This is, on the one hand, clearly a disadvantage because no complete information about the composition of the sample is available. On the other hand, this may also be considered as an advantage because the complexity of the spectra is considerably reduced. These aspects are discussed in this article. It is shown in a 3-h experiment that different PL classes are detectable with different sensitivities.
_
Hazards Great care should be taken if CHCl3 is handled because this solvent is suspected to cause cancer. Methanol is a flammable liquid and is irritating to the skin, eyes, and respiratory tract. Methanol may also be fatal if swallowed. Contact with 2,5dihydroxybenzoic acid (DHB) that is used as MALDI matrix should be also avoided. The toxicological properties of the different PLs have not been fully investigated; however, they may cause eye, skin, respiratory, and digestive irritation. Protective clothing (as well as goggle and gloves) should be worn at all times during the sample preparation step. Extraction of Tissues It is nearly impossible to extract lipids quantitatively (7). Thus, it cannot be claimed here that complete extraction of all lipid classes is achieved. Nevertheless, the obtained lipid yield by using extraction with organic solvents is sufficient for subsequent characterization, as we focus on the most abundant PL classes. A more detailed description of the extraction process, sample preparation, and the MALDI-TOF MS measurements is provided in the supporting information. Results To obtain experience with the appearance of the MALDI mass spectra of lipids, some isolated lipids are initially investigated by MALDI-TOF MS. In Figure 1, the positive ion MALDI-TOF mass spectra of triolein, 1-palmitoyl-2-oleoyl-sn-phosphatidylethanolamine (POPE), and 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC) are shown. All spectra were recorded in the presence of 2,5-dihydroxybenzoic acid (DHB), an established acidic matrix for lipids (10). The spectrum of the POPC (Figure 1C) is simple and both detectable peaks at m/z = 760.6 and 782.6 can be explained as the Hþ and the Naþ adduct, respectively. No fragmentation of this analyte occurs in the observed mass range. The spectrum of the POPE differs (Figure 1B) from that of POPC, although there is only a slight difference in the headgroup structures between the molecules. The monosiotopic weight of POPE is 717.5 and, thus, the peaks at m/z = 718.5 and 740.5 can be explained as the Hþ and the Naþ adducts, respectively. However, there is one additional peak at m/z = 762.5 that corresponds to the ion POPE Hþ þ 2 Naþ. The reason why such an ion is not detected in the
_
r 2011 American Chemical Society and Division of Chemical Education, Inc. pubs.acs.org/jchemeduc Vol. 88 No. 4 April 2011 10.1021/ed1004905 Published on Web 02/01/2011
_
Journal of Chemical Education
503
In the Laboratory
Figure 1. Positive ion MALDI-TOF mass spectra of selected lipids: (A) triolein, (B) POPE, and (C) POPC. Lipids (1 mg/mL in CHCl3) were mixed (1:1 v/v) prior to deposition onto the MALDI target with 0.5 M DHB in CH3OH.
case of the POPC stems from the different headgroups: The quaternary ammonia group of POPE possesses acidic properties and can, thus, show exchange with other ions such as Naþ. In contrast, the quaternary ammonia group of the POPC is not acidic and, thus, no exchange will take place. Also note that there are small peaks at m/z = 756.5 and 778.5 that indicate the presence of Kþ ions. Regarding the affinity of PLs to alkali metal ions, there are no marked differences and different ions are bound according to their concentrations (11). It is also remarkable that the POPE provides, in contrast to the POPC, two peaks (at m/z = 577.5 and 603.5) that correspond to a loss of the headgroup (cf. structure in Figure 1B). The reasons for this discrepancy are unknown; however, a similar mechanism suggested to explain the mass spectra of triacylglycerols (TAG) (12) may also be valid in the case of the POPE (13). This will be discussed in the context of the triolein (Figure 1A). Considering the spectrum of the triolein (its monoisotopic weight is 884.7), it is evident (Figure 1A) that there is exclusively the Naþ adduct (m/z = 907.7), but the expected Hþ adduct (m/z = 885.7) is not detected. Although this phenomenon has been known for many years (5), a convincing explanation was only recently provided. To explain these data, it is necessary to emphasize that MS detects only ions that are sufficiently stable to “survive” the flight path without fragmentation. For the majority of MALDI-TOF devices, this corresponds to a few microseconds. Gidden et al. (12) have shown that the Hþ adducts (that are actually generated from TAG if an acidic matrix is used) are not sufficiently stable and decompose during the flight to the mass analyzer by losing one sodium salt of one fatty acyl residue. 504
Journal of Chemical Education
_
Vol. 88 No. 4 April 2011
_
This leads to the signal at m/z = 603.5. In contrast, the stability of the Naþ adducts is much higher allowing them to enter the mass analyzer without fragmentation (12). Such stability considerations are, thus, important and essential for the successful interpretation of a mass spectrum. Although a couple of different lipid extracts (see below) are discussed in this article, the extract from the avocado is unique because it yields nearly exclusively TAG, whereas the PL content is extremely low. This is evident from the videodensitometric image (Figure 2) of a developed TLC plate containing the relevant samples. Each of the isolated compounds (POPC, POPE, triolein) gives a single spot. In contrast, the egg yolk and the liver extract indicate the presence of more than one lipid class. Although we do not discuss these PLs in detail but focus on the both most abundant ones, PC and PE, a more detailed assignment of the residual classes is shown on the left side of Figure 2. The TLC is also used in a preparative way to isolate some selected lipid classes. Thus, only dyes that do not result in changes of the molecular weights of the lipids can be used. Primuline (14) fulfils this requirement. However, this dye binds with different efficiencies to lipids, and thus, quantitative data cannot be obtained from the spot intensities on the TLC plate. Note that the avocado extract gives just a single spot near to the solvent front corresponding to TAG. The exclusive presence of TAG is also clearly reflected in the MALDI-TOF mass spectrum of the avocado extract (Figure 3). The peak at m/z = 907.7 (cf. the emphasized mass region) has been already assigned in trace A of Figure 1 and corresponds to triolein. Of course, this detailed assignment can actually not be made and an isomer, for instance, a replacement of two oleoyl
pubs.acs.org/jchemeduc
_
r 2011 American Chemical Society and Division of Chemical Education, Inc.
In the Laboratory
residues by one stearoyl and one linoleoyl residue would also fit the observed m/z ratio. This problem can be overcome by MS/ MS techniques (15) but is outside the scope of this article. It is also evident that the peak at m/z = 881.7 shows a characteristic mass difference of 26 amu with reference to the peak at m/z = 907.7 and this characteristic mass difference corresponds to the exchange of one oleoyl (18:1) by one palmitoyl (16:0) residue. Further information about the MALDI-TOF MS characterization of TAG is available in refs (15) and (16). The positive ion MALDI-TOF mass spectra of a total tissue extract compares with PL fractions isolated by means of TLC (cf. Figure 2). Experimental details are available in the supporting information as well as in ref 7. Although tissues contain a lot of
Figure 2. Videoimage of a TLC plate (containing the isolated lipids or the indicated tissue extracts) subsequent to primuline staining (14): Cho, cholesterol; LPC, lyso-phosphatidylcholine; LPE, lyso-phosphatidylethanolamine; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PI, phosphatidylinositol; SM, sphingomyelin; TAG, triacylglycerol. Note that there are some weak bands in the liver extract that were tentatively assigned to glycolipids but are not characterized in detail. The small quantity of LPC in the POPC sample should be regarded as an impurity (17). Also note that the Rf values of TAG and cholesterol are virtually identical.
different lipid classes, we focus exclusively on PC and PE because these both PL classes are normally the dominating species in tissue extracts (18). In Figure 4, the spectrum of the total extract from hen egg yolk is shown and compared with the spectra of the purified PC and PE fractions. Considering the total extract, it is evident that POPC (m/z = 760.6 and 782.6) is the most abundant species (accompanied by smaller quantities of PLPC, m/z = 758.6 and 780.6) and this agrees favorably with the spectrum recorded from the isolated PC fraction (Figure 4B). The reason of the much higher intensity of the Naþ adduct (m/z = 782.6) in comparison to the Hþ adduct (m/z = 760.6) subsequent to the TLC separation is the use of physiological saline (154 mmol/L NaCl) to reelute the lipids from the silica gel (cf. the available supporting information for more details). Therefore, there is a higher content of Naþ ions in the trace in Figure 4B and this is reflected by the enhanced intensity of the Naþ adducts (11). Irrespective of this difference, both spectra are virtually identical. However, it is evident that there is also a significant PE moiety (Figure 4C) that is easily seen on the TLC plate (cf. Figure 2) but is exclusively detectable subsequent to separation from the PC that clearly dominates the positive ion MALDI-TOF mass spectra of the total extract (20). A more detailed assignment of all peaks detected in the individual PL fractions is available in the supporting information. We only consider the peaks at m/z = 768.5, 790.5, and 812.5 that are caused by PE 16:0/20:4, which is the most abundant PE species of the egg yolk. The positions of these peaks are marked by the dotted lines in Figure 4 and it is obvious that these peaks are not detectable in the total egg yolk extract. If one would consider exclusively the spectrum of this extract, one would (incorrectly) conclude that the egg yolk contains no lipids beside PC (20). A few years ago, the MALDI-TOF MS detectabilities of the individual PL classes were compared (21) and it was shown that PC is about 50 times more sensitively detectable than PE, that is, the reported detection limits were about 20 pg and 1 ng, respectively (21). Although detection limits are significantly dependent on the used matrix (the authors used DHB (21) as we here also do), this is a clear indication that detectabilities of PC and PE are significantly different (9).
Figure 3. Positive ion MALDI-TOF MS of an organic avocado extract recorded in the presence of DHB as matrix. Peaks of the intact TAG are emphasized in the upper part of the figure. All peaks are marked according to their m/z ratios. The typical DHB matrix peak at m/z = 551.0 (10) is labeled by an asterisk.
r 2011 American Chemical Society and Division of Chemical Education, Inc.
_
pubs.acs.org/jchemeduc
_
Vol. 88 No. 4 April 2011
_
Journal of Chemical Education
505
In the Laboratory
Figure 4. Positive ion MALDI-TOF mass spectra of an organic extract obtained according to the method by Bligh and Dyer (19) of hen egg yolk: (A) the mass spectrum of the total extract, (B) the PC fraction, and (C) the PE fraction. All spectra were recorded with DHB as the matrix. Separation of lipids was performed by TLC prior to MS analysis. The dotted lines indicate the peaks of the most abundant PE species, PE 16:0/20:4.
Figure 5. Positive ion MALDI-TOF mass spectra of an organic extract (obtained according to the method by Bligh and Dyer) of bovine liver: (A) the mass spectrum of the total extract, (B) the PC fraction, and (C) the PE fraction. All spectra were recorded with DHB as matrix. Separation of lipids was performed by TLC prior to MS analysis. Although some further TLC fractions could be obtained, only the most relevant lipid classes are shown here. The dotted lines indicate the peaks of the most abundant PE species PE 16:0/20:4.
Lipids from egg yolk are often used as model compounds owing to their simple availability. Although the lipid composition of egg yolk may vary, a coarse composition of the egg yolk lipids can be easily provided (18, 22): Although PC contributes about 75%, the contribution of the PE is about 17%; that is, there is much more PC than PE. Thus, one might argue that the effects demonstrated in Figure 4 are not exclusively caused by differences 506
Journal of Chemical Education
_
Vol. 88 No. 4 April 2011
_
in the detectabilities of the different PL but are also stemming from the higher PC content of the hen egg yolk. Therefore, in addition to the hen egg yolk extract, an extract of bovine liver was also investigated because the liver has a higher PE content in comparison to the egg yolk. Note that these compositional data were not taken from the chromatogram (Figure 2) but were determined by 31P NMR (data not shown) (23). This was necessary because
pubs.acs.org/jchemeduc
_
r 2011 American Chemical Society and Division of Chemical Education, Inc.
In the Laboratory
the TLC spot intensities are only semiquantitative interpretable because the primuline dye is not bound to all PL classes to the same extent. Another problem is the application of the dye by spraying may result in inhomogeneities. Thus, the aim of the TLC was primarily the determination of different relevant PL spots. In Figure 5, the positive ion MALDI MS of the total extract from bovine liver is shown and compared with the spectra of the PC and the PE fraction, both obtained by TLC separation of the total extract. Comparing the total extract (Figure 5A) with the PE fraction (Figure 5C), it is evident that PE species are only detectable with very low sensitivities (cf. the peaks at m/z = 768.5, 790.5, and 812.5 marked by vertical dotted lines). Therefore, PE is not as sensitively detectable even if it is present in comparable quantities as the PC. A more detailed assignment of all the compounds detected in the different fractions is available in the supporting information. Summarizing, it is obvious from the shown examples that not all compounds are detected to the same extent if mixtures are analyzed. This does not only hold for lipids, but is valid for virtually all compounds, for instance, peptides and carbohydrates. Therefore, previous separation is required if detailed compositional data are required. Educational Value We have shown that the mass spectrometric analysis of mixtures must be regarded with great caution because it is unlikely that all compounds, in particular if they differ in the presence of charged groups, are detected to the same extent. In the worst case, selected compounds are not detectable at all. This is an important “take home message” for advanced students of chemistry or biochemistry where the knowledge of basics of MS and particularly soft-ionization methods such as ESI or MALDI are regarded as indispensable. It is particularly important to tell these students that this is valid for all substances, equally if lipids, proteins, or carbohydrates are of interest. Acknowledgment This work was supported by the German Research Council (DFG Schi 476/12-1 and FU 771/1-1). Additionally, this publication was supported by LIFE: Leipzig Research Center for Civilization Diseases, University of Leipzig. LIFE is funded by means of the European Union, by the European Regional Development Fund (ERDF) and by means of the Free State of Saxony within the framework of the excellence initiative. Literature Cited 1. Fuchs, B.; Schiller, J. Subcell. Biochem. 2008, 49, 541–565.
r 2011 American Chemical Society and Division of Chemical Education, Inc.
_
2. Murphy, R. C.; Hankin, J. A.; Barkley, R. M. J. Lipid Res. 2009, 50 (Suppl.), S317–322. 3. Peterson, B. L.; Cummings, B. S. Biomed. Chromatogr. 2006, 20, 227–243. 4. Pulfer, M.; Murphy, R. C. Mass Spectrom. Rev. 2003, 22, 332–364. 5. Schiller, J.; S€uß, R.; Arnhold, J.; Fuchs, B.; Leßig, J.; M€uller, M.; Petkovic, M.; Spalteholz, H.; Zschörnig, O.; Arnold, K. Prog. Lipid Res. 2004, 43, 449–488. 6. Hillenkamp, F.; Peter-Katalinic, J. MALDI MS;A Practical Guide to Instrumentation. Methods and Application, 1st ed.; Wiley-VCH: Weinheim, 2007. 7. Fuchs, B.; Nimptsch, A.; S€ uß, R.; Schiller, J. Methods Mol. Biol. 2009, 579, 103–125. 8. Mormann, M.; Bashir, S.; Derrick, P. J.; Kuck, D. J. Am. Soc. Mass Spectrom. 2000, 11, 544–552. 9. Petkovic, M.; Schiller, J.; M€ uller, M.; Benard, S.; Reichl, S.; Arnold, K.; Arnhold, J. Anal. Biochem. 2001, 289, 202–216. 10. Schiller, J.; S€uß, R.; Fuchs, B.; M€uller, M.; Petkovic, M.; Zschörnig, O.; Waschipky, H. Eur. Biophys. J. 2007, 36, 517–527. 11. Schiller, J.; Arnhold, J.; Benard, S.; M€uller, M.; Reichl, S.; Arnold, K. Anal. Biochem. 1999, 267, 46–56. 12. Gidden, J.; Liyanage, R.; Durham, B.; Lay, J. O., Jr. Rapid Commun. Mass Spectrom. 2007, 21, 1951–1957. 13. Fuchs, B.; Schiller, J. Eur. J. Lipid Sci. Technol. 2009, 111, 83–98. 14. White, T.; Bursten, S.; Frederighi, D.; Lewis, R. A.; Nudelman, E. Anal. Biochem. 1998, 10, 109–117. 15. Pittenauer, E.; Allmaier, G. J. Am. Soc. Mass Spectrom. 2009, 20, 1037–1047. 16. Schiller, J.; S€ uß, R.; Petkovic, M.; Arnold, K. J. Food Lipids 2002, 9, 185–200. 17. Hernandez-Caselles, T.; Villalaín, J.; Gomez-Fernandez, J. C. J. Pharm. Pharmacol. 1990, 42, 397–400. 18. Fuchs, B.; Schiller, J.; S€ uß, R.; Sch€urenberg, M.; Suckau, D. Anal. Bioanal. Chem. 2007, 389, 827–834. 19. Bligh, E. G.; Dyer, W. J. Can. J. Biochem. Physiol. 1959, 37, 911–917. 20. Schiller, J.; S€uß, R.; Fuchs, B.; M€ uller, M.; Zschörnig, O.; Arnold, K. Front. Biosci. 2007, 12, 2568–2579. 21. Gellermann, G. P.; Appel, T. R.; Davies, P.; Diekmann, S. Biol. Chem. 2006, 387, 1267–1274. 22. Rhodes, D. N.; Lea, C. H. Biochem. J. 1957, 65, 526–533. 23. Schiller, J.; M€uller, M.; Fuchs, B.; Arnold, K.; Huster, D. Curr. Anal. Chem. 2007, 3, 283–301.
Supporting Information Available Description of the extraction process; sample preparation; and the MALDI-TOF MS measurements. This material is available via the Internet at http://pubs.acs.org.
pubs.acs.org/jchemeduc
_
Vol. 88 No. 4 April 2011
_
Journal of Chemical Education
507
