Progress in Lipid Research

Transcription

1 Progress in Lipid Research 49 (2010) Contents lists available at ScienceDirect Progress in Lipid Research journal homepage: Review An update of MALDI-TOF mass spectrometry in lipid research Beate Fuchs, Rosmarie Süß, Jürgen Schiller * University of Leipzig, Medical Department, Institute of Medical Physics and Biophysics, Härtelstraße 16-18, D-04107, Germany article info abstract Article history: Received 25 April 2010 Received in revised form 29 June 2010 Accepted 1 July 2010 Keywords: MALDI-TOF MS Lipids Phospholipids Matrix Although matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) often but not exclusively coupled with a time-of-flight (TOF) mass analyzer is primarily established in the protein field, there is increasing evidence that MALDI MS is also very useful in lipid research: MALDI MS is fast, sensitive, tolerates sample impurities to a relatively high extent and provides very simple mass spectra without major fragmentation of the analyte. Additionally, MALDI MS devices originally purchased for proteomics can be used also for lipids without the need of major system alterations. After a short introduction into the method and the related ion-forming process, the MALDI mass spectrometric characteristics of the individual lipid (ranging from completely apolar hydrocarbons to complex glycolipids with the focus on glycerophospholipids) classes will be discussed and the progress achieved in the last years emphasized. Special attention will be paid to quantitative aspects of MALDI MS because this is normally considered to be the weak point of the method, particularly if complex lipid mixtures are to be analyzed. Although the detailed role of the matrix is not yet completely clear, it will be also explicitly shown that the careful choice of the matrix is crucial in order to be able to detect all compounds of interest. Two rather recent developments will be highlighted: Imaging MS is nowadays widely established and significant interest is paid in this context to the analysis of lipids because lipids ionize particularly well and are, thus, more sensitively detectable in tissue slices than other biomolecules such as proteins. It will also be shown that MALDI MS can be very easily combined with thin-layer chromatography (TLC) allowing the spatially-resolved screening of the entire TLC plate and the detection of lipids with a higher sensitivity than common staining protocols. Ó 2010 Elsevier Ltd. All rights reserved. Contents 1. Introduction Soft ionization mass spectrometric methods Fundamentals of MALDI mass spectrometry The role of the matrix some practical considerations Typical MALDI matrices Inorganic MALDI matrices Abbreviations: 9-AA, 9-aminoacridine; amu, atomar mass unit; APCI, atmospheric pressure chemical ionization; CE, cholesteryl ester; CHCA, a-cyano-4-hydroxycinnamic acid; CID, collisionally-induced dissociation; DAG, diacylglycerols; DE, delayed extraction; DESI, desorption electrospray; DGDG, digalactosyl-diacylglycerol; DHB, 2,5- dihydroxybenzoic acid; DIOS, desorption/ionization on silicon; DMAN, 1,8-bis-(dimethylamino)-naphthalene; EI, electron ionization; Er:YAG, erbium-doped yttrium aluminium garnet; ESI, electrospray ionization; FAB, fast atom bombardment; FD, field desorption; FI, Field Ionization; FT, Fourier Transform; GALDI, graphite-assisted laser desorption/ionization; GC, gas chromatography; GPL, glycerophospholipid; HPLC, high-performance liquid chromatography; HPA, hydroxy-picolinic acid; ILS, ionic liquids; IP, ionization potential; IR, infrared; LD, laser desorption; LOD, level of detection; LOQ, level of quantification; LPA, lysophosphatidic acid; LPC, lyso(monoacyl)- phosphatidylcholine; LPL, lyso(monoacyl)-phospholipid; MALDI, matrix-assisted laser desorption and ionization; MGDG, monogalactosyl-diacylglycerol; MS, mass spectrometry; MTPFPP, meso-tetrakis(pentafluorophenyl)porphyrin; m/z, mass over charge; Nd:YAG, neodymium-doped yttrium aluminium garnet; NMR, nuclear magnetic resonance; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP 2, phosphatidylinositol- 4,5-bisphosphate; PL, phospholipid; PLA 2, phospholipase A 2 ; PNA, para-nitroaniline; PPI, (poly-)phosphoinositides; PS, phosphatidylserine; PSD, post source decay; PVDF, polyvinylidene difluoride; RF, retardation factor; ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; SA, sinapic acid; SCX, silica gel cation exchanger; SIMS, secondary ion MS; SM, sphingomyelin; S/N, signal to noise; sn, stereospecific numbering; SQDG, sulfoquinovosyl-diacylglycerol; TAG, triacylglycerol; TCA, trans-4-hydroxy- 3-methoxycinnamic acid; TFA, trifluoroacetic acid; THA, trihydroxy-acetophenone; TLC, thin-layer chromatography; TOF, time-of-flight; UV, ultraviolet. * Corresponding author. Tel.: ; fax: address: juergen.schiller@medizin.uni-leipzig.de (J. Schiller) /$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved. doi: /j.plipres

2 B. Fuchs et al. / Progress in Lipid Research 49 (2010) A survey of lipids so far investigated by MALDI-TOF MS Hydrocarbons and wax esters Plant pigments Flavonoids and carotenoids Free fatty acids Cholesterol and cholesteryl esters Sphingolipids Glycerolipids Di- and triacylglycerols Glycoglycerolipids Glycerophospholipids Zwitterionic glycerophospholipids (PC and PE) Acidic glycerophospholipids Analysis of lipid mixtures Separation of the individual lipid classes prior to analysis Glyco- and sphingolipids Glycerophospholipids MALDI MS imaging Summary Acknowledgments References Introduction We are currently living in an omics time period [1]. Although this list is surely not complete, there were already proteomics, genomics, lipidomics, metabonomics, metagradomics, metallomics and rather recently interactomics. As these approaches provide an immense amount of data, bioinformatics is an indispensable counterpart leading to systems biology initiatives [2] that may help to get further insights into complex metabolic networks of biological systems. As this review is dedicated to lipids and their analysis, a short definition of lipidomics is necessary: according to a recently provided definition [3], lipidomics can be defined as the full characterization of lipid molecular species and of their biological roles with respect to expression of proteins involved in lipid metabolism and function, including gene regulation. The combination of all these complex aspects is clearly a very challenging task, but the first step is obviously the qualitative and quantitative knowledge of the lipid composition of an unknown sample. Many omics applications are based on the application of mass spectrometry (MS) and would not have been possible without the significant progress achieved in this field during the last decades. MS is an incredible powerful method and may be regarded as an indispensable tool in physics, chemistry, biochemistry and (increasingly) medicine and particularly clinical diagnosis [4]. The history of MS began more than one century ago and was initially related to the discovery of the different isotopes of the chemical elements [5] and, thus, primarily a task of physicists. For instance, Thompson was awarded the Nobel Prize (of Physics) in 1906 for his work on Conduction of Electricity through Gases. In contrast, the history of the success of MS in biology and life sciences is much shorter and directly related to the discovery of new and gentle ionization methods: over many decades electron ionization (EI) was exclusively available as ionization method [4]. EI makes use of fast electrons to ionize the analyte molecules in the gas phase by the removal of one electron. Although this technique was (and still is) unequivocally suitable for the investigation of small and/or volatile compounds, EI normally fails if larger molecules with low volatilities are to be analyzed: although it could be shown that the molecular ion (M? M + +e ) of phospholipids is basically detectable if EI is used for ionization of the sample, the obtained results were not very convincing [6] and fragment ions are much more abundant in comparison to M + making this technique less suitable for the analysis of mixtures. Therefore, the prime application of EI MS in the context of lipids is nowadays normally the analysis of free fatty acids that can be obtained by saponification of lipids. This also holds for oxidation products derived from free fatty acids and their metabolic products such as thromboxanes or leukotrienes [7]. GC/MS is even nowadays a highly established and widely used method for the quantitative analysis of relatively apolar compounds. Unfortunately, sample preparation is quite cumbersome and time-consuming. Because of these disadvantages, the invention of soft-ionization methods, enabling the analysis of molecules refractive to EI, must be regarded as a real milestone in the history of modern mass spectrometry [8] Soft ionization mass spectrometric methods The differentiation between hard and soft ionization MS methods is a matter of philosophy and depends to a significant extent on the analyte of interest and its properties. However, all modern ionization methods may be regarded as softer than EI [9]. Methods enabling the ionization of compounds refractive to electron collision ionization comprise gas phase ionization techniques such as chemical ionization (CI) that was discovered in 1913 by Thompson [10], field desorption (FD) and field ionization (FI), methods based on particle bombardment such as fast atom bombardment (FAB) or secondary ion MS (SIMS) [11]. Each of these ionization methods has its individual strengths and weaknesses, but the application of these methods is normally limited to special problems or selected analytes [4]. Only chemical ionization (often in combination with atmospheric pressure, APCI) is used to a higher extent for the analysis of lipids. For a recent review of applications of CI in lipid research see [12], while a survey of the advantages and drawbacks of the different ionization techniques is available in [13]. Today, electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) MS are most often used and the majority of commercially available MS devices is equipped with one (or maybe even both) of these ion sources. As a maximum of information is obviously achievable if both techniques are combined, they should be regarded as complementary but not as competitive. The importance of these both ionization methods was obviously also the reason why the inventors of ESI and MALDI, i.e. Fenn and coworkers [14] and Tanaka and coworkers [15], respectively, shared the Nobel Prize for Chemistry in 2002 [16].

3 452 B. Fuchs et al. / Progress in Lipid Research 49 (2010) We will not provide here a comprehensive survey of the fundamentals of ESI MS (for reviews dedicated to ESI MS analysis of lipids see [17,18]), but will focus on applications of MALDI MS and the related methodology. A review with a similar topic was already published by our group in 2004 in Progress in Lipid Research [19]. However, many important improvements regarding potential applications, matrix optimization, quantitative data analysis and more detailed characterization (particularly by MS/MS) of the different lipid classes have been achieved since that time and an update of our previous review seemed necessary. In our opinion the most important developments can be summarized as follows: 1. Although details of ion generation are still not yet completely understood, some progress in understanding this important aspect has been achieved. This is very important because further theoretical insights will help to improve the performance of the MALDI method. A comprehensive survey of theoretical aspects is available in [20]. 2. Some new and improved matrix compounds were introduced that provide either higher sensitivities or higher reproducibilities than previously used matrices [21]. We will emphasize here that some lipid-derived compounds are exclusively detectable if the most appropriate matrix is used. 3. The coupling between MALDI MS and chromatographic separation techniques is unequivocally a hot topic of current research. This particularly concerns the combination between MALDI MS and thin-layer chromatography (TLC) that helps to overcome many problems related to mixture analysis in a simple but elegant way [22]. 4. MS imaging is nowadays an established method to monitor the distribution of certain molecules within a tissue. As lipids ionize particularly well, they are very easily detectable under these conditions and this fact has pushed the interest in lipid analysis by MALDI MS considerably [23]. The continuously increasing interest in MALDI MS as well as its applications in lipid analysis is clearly reflected by the data given in Fig. 1. It must be explicitly stated that we do not want to suggest MAL- DI MS as the best method of lipid analysis. However, during the last centuries there were a lot of omics initiatives and many MALDI devices were purchased particularly for protein and peptide analysis. It is our aim to convince the reader that these instruments are not exclusively useful in the proteomics field, but the same instruments can be readily used for the analysis of lipids. There is surely no need to buy an additional device but the available MALDI devices can be used without the need of major alterations! 1.2. Fundamentals of MALDI mass spectrometry A detailed survey of methodological aspects of MALDI MS is available in the excellent book by Franz Hillenkamp and Jasna Peter-Katalinić [20]. Therefore, theoretical fundamentals of MALDI MS will be only shortly discussed in this review. However, a short introduction is necessary for the less experienced reader. MALDI MS is based on the utilization of a matrix that initially absorbs the energy of the laser and mediates the generation of ions. Although inorganic compounds such as graphite [24] or metal oxide particles [25] may be also applied as matrix, small organic molecules are used in the majority of cases and will be, thus, nearly exclusively discussed here. Of course, the suitability of a certain compound as matrix is determined by the type of the laser and its emission wavelength. Although many different lasers, including Nd:YAG (k = 355 or 266 nm) [26], excimer [27] or CO 2 lasers [28] were already successfully applied, in the lipid field N 2 lasers are primarily used. Although IR lasers provide some advantages (see below), we will focus here nearly exclusively on UV lasers with an emission wavelength of 337 nm because the majority of commercially available MALDI devices are equipped with this laser type. This is the reason why the majority of the so far available lipid data were obtained with UV lasers. A more comprehensive discussion of the role of the matrix is available in [29]. When the pulsed laser beam hits the sample (normally cocrystals of the matrix and the analyte), its energy is primarily absorbed by the matrix that is present in a vast excess over the analyte (a ,000-fold excess of the matrix is typically used). Consequently, the matrix is vaporized, carrying intact analyte molecules into the vapor phase. A simplified schema of the processes occurring in a typical MALDI-TOF mass spectrometer is shown in Fig. 2 [19]. During the expanding process of this gas cloud, ions (e.g. H + and Na + ) are exchanged between the matrix and the analyte, leading to the formation of charged analyte molecules. These analyte ions are called adducts or quasimolecular ions (sometimes the term pseudomolecular ions may be also found although the use of this term is discouraged by the IUPAC). Beside cation generation, anions can also be generated by abstracting H + or Na + from the analyte. The ratio between the cation and the anion yield is determined by the (gas phase) acidities of the analyte and the matrix [20] and fundamentals of the ion formation process were recently comprehensively reviewed by Zenobi and Kochenmuss [30,31]. Recording positive-ion mode spectra is much more common and it even seems (although not yet investigated in detail) that many MALDI mass spectrometers detect negative ions less sensitively than positive ions. Fig. 1. Number of scientific papers containing the term MALDI or matrix-assisted (a) and papers that contain additionally the term lipid or phospholipid (b). All data were taken from the Web of Science database.

4 B. Fuchs et al. / Progress in Lipid Research 49 (2010) The most important difference between molecular (radical) ions (generated in conventional EI mass spectra) and quasimolecular ions is the observed mass. As molecular ions are generated by the abstraction of one electron (the mass of which can be neglected for the majority of applications because it accounts for just about two-tenths of a percent of the mass of a proton) from the analyte, the mass of the generated ion (or the observed m/z ratio) corresponds to the mass of the analyte. In contrast, quasimolecular cations are generated by the addition of a cation to the analyte. Therefore, the mass of the quasimolecular ion is characteristically higher (normally 1 amu or 23 amu corresponding to the mass of H + or Na +, respectively) in comparison to the analyte molecule [32]. Despite its profound importance, the process of ion generation is so far only poorly understood and many papers are currently dealing with this important topic. For instance, one recent issue of European Journal of Mass Spectrometry (2006, Volume 12, Issue 6) was exclusively dedicated to the topic Mechanisms of MALDI. Despite this obvious lack of knowledge, it is sure that singlycharged ions are primarily generated [33] and, therefore, the actual measured quantity, the mass-to-charge ratio (m/z) may be replaced directly by the monoisotopic mass of the analyte molecule plus or minus the mass of the ion required to generate a charge. The use of Thompson [Th] as unit of the mass over charge ratio was suggested nearly 20 years ago [34] but is not yet widely established. A detailed discussion of this aspect is available in [35]. After being formed, ions are accelerated in an electric field (typically of the order of 20 kv). After passing a charged grid, the ions are drifting freely over a field-free space where mass separation is achieved: low mass ions arrive at the detector in a shorter time than high mass ions [13]. This is the most simple, linear geometry of a TOF analyzer that is normally used for the analysis of larger molecules because in this case high resolution is less important than high sensitivity. Resolution and peak widths may be improved by using a reflectron [20]. The reflectron enlarges the flight path and helps to compensate differences in the initial velocities of the ions during the ablation process. All spectra shown in this review were recorded on a MALDI-TOF device equipped with a reflectron. Using commercial MALDI-TOF devices with reflectron configuration, mass resolutions of about 10,000 and mass accuracies higher than about 30 ppm can be routinely achieved. This is rather poor in comparison to the best available mass spectrometers nowadays which provide mass accuracies lower than 1 ppm and also much higher resolutions. Such high quality mass spectra are desirable in the context of proteomics, where an increased mass accuracy helps to unequivocally assign unknown (tryptic) peptides because the number of hits upon database searching is significantly reduced [36] if the mass can be provided with higher accuracy. In the context of lipids, however, very high mass accuracies do not really provide a much larger extent of information because the smallest mass difference within one given lipid class is 2 amu, i.e. one double bond. We will show below that there are some simple chemical/biochemical methods (addition of certain salts or enzymatic digest) to overcome problems related to potential signal overlap and a MALDI device with a high resolving power is not absolutely needed for routine lipid analysis. It should be noted that there is no absolute need to combine a MALDI ion source with a TOF mass analyzer. However, as MALDI is used in the majority of cases for the detection of rather large molecules, the TOF detector is very popular because it has a nearly unlimited mass range [20]. An additional reason is the pulsed (not continuous) ion generation of MALDI that is most suitable for the TOF mass analyzer. A more comprehensive survey of typical mass analyzers as well as their individual advantages and drawbacks is available in [13,20]. 2. The role of the matrix some practical considerations Although there were already different attempts to predict the suitability of an unknown chemical compound as MALDI (UV) matrix by theoretical considerations, most matrices were (and actually are) found by accident or by a try and error approach. A more detailed discussion of these aspects is beyond the scope of this review and we will focus exclusively on some selected practical aspects. A good MALDI UV matrix in the practical sense is characterized by the following properties: 1. A suitable matrix should provide an excellent signal-to-noise (S/ N) ratio for the peaks of the analyte of interest, i.e. a high sensitivity should be achievable. 2. The matrix should provide a high absorbance at the emission wavelength of the laser. Thus, the required laser fluence should be as low as possible because enhanced laser fluence leads to enhanced analyte fragmentation and/or poorly resolved mass spectra. 3. An optimum matrix is characterized by low background (i.e. the signals of the matrix should be very small) in order to avoid interferences between the matrix and the analyte ions. It is important to note that oligomers of the matrix are often generated in the gas phase. Therefore, most matrices provide signals at m/z ratios much higher than their actual molecular weight! 4. Finally, an ideal matrix should provide only a single adduct of the analyte and exhibit a weak tendency to cluster formation because analyte matrix clusters may seriously complicate data analysis [37]. Reflector Detector mass to charge (m/z) Matrix Sample Plate Nitrogen Laser (337 nm) Charged Grid Reflector ("electrostatic Mirror") Linear Detector Sample Target High Voltage Heavy IonsLi ght Ions Field-free time-of-flight mass to charge (m/z) Fig. 2. Schema of the processes occurring during the MALDI-TOF ionization process in the mass spectrometer (for details see text). The influence of the detection using the linear and reflector mode is emphasized in the figure. Reprinted with modification and permission from Elsevier [19].

5 454 B. Fuchs et al. / Progress in Lipid Research 49 (2010) As the vast majority of commercially available MALDI devices are equipped with nitrogen lasers, the focus of this review will be on matrices compatible with N 2 lasers (337 nm), whereas infrared lasers and the corresponding matrices (such as glycerol or succinic acid) will be treated only briefly. There are basically five typical requirements that must be met by a powerful MALDI matrix and some selected UV MALDI matrix compounds are shown in Fig. 3. (a) The matrix must exhibit a strong absorption at the laser emission wavelength, i.e. in the case of a UV laser normally at 337 nm. This is the reason why nearly all common organic matrices contain an aromatic ring system with delocalized p electrons. Inorganic matrices such as graphite or metal salts will only be shortly discussed here because they are scarcely applied to the field of lipid analysis so far [38]. As expected, the ionization efficiency increases if the absorption coefficient of the matrix at the laser wavelength increases and this is one reason why among the different isomers of dihydroxybenzoic acids (DHB), only the 2,5 isomer that provides the most marked absorption at 337 nm (but not the other isomers) is commonly used as MALDI matrix [39]. Although the solution UV absorption of the matrix is determined in many studies, it must be emphasized that exclusively the UV absorption in the solid state determines the absorption properties under MALDI conditions. Absorptions determined in the liquid and in the solid state may be significantly different [39]. (b) The energy absorption and the subsequent evaporation of the matrix must lead to the generation of ions from the analyte of interest. This is often believed to be the reason for the use of carboxylic acids (many matrices are derived from cinnamic or benzoic acids) as matrix compounds because organic acids are acidic and, thus, capable of triggering the generation of H + adducts. As sodium is an ubiquitous element, however, there are normally also Na + adducts in addition to H + adducts. In addition to the enhanced generation of H + adducts, there is another practical reason to use carboxylic acids: due to the presence of the aromatic ring system, typical MALDI matrices are well soluble in organic solvents. As MALDI is particularly used for the analysis of polar molecules that are only scarcely soluble in organic solvents, water is one important constituent of the most common solvent systems: the presence of polar groups (such as carboxylic acids) increases the solubility of the matrix in polar solvents [13]. (c) The matrix should be stable under high vacuum conditions. Although this sounds trivial, there are many potentially useful MALDI matrices that do not or not sufficiently fulfill this criterion. As most MALDI devices make use of high vacuum conditions (normally about bar) many compounds tend to sublime. The loss of the matrix under high vacuum conditions may be one important reason why MALDI mass spectra show time-dependent changes [40]. (d) The matrix should isolate the generated ions and prevent the generation of analyte clusters, for instance, dimer formation. This is the prime reason why a significant excess of the matrix over the analyte is normally required in order to obtain optimum results. Additionally, the laser fluence should be kept as high as needed but as low as possible because matrix analyte clusters are increasingly generated at elevated laser fluences [19]. (e) The matrix and the analyte should give homogenous cocrystals. Improvement of homogeneity is clearly science of its own and depends beside the analyte, the matrix and the solvent system on the method of sample preparation. This important topic would be a review of its own and has been reviewed elsewhere [41] but with the focus on polar molecules. Briefly, the simpler the sample preparation method, the lower is the homogeneity of the matrix/analyte mixture. The most frequently used dried droplet method, i.e. the successive deposition of matrix and analyte are normally not suitable if any quantitative data have to be derived from the mass spectra. In contrast, the more sophisticated sample preparation by electrospray deposition provides a much more homogeneous matrix/analyte mixture and normally permits quantitative data evaluation. Although MALDI targets made from different metals (e.g. aluminum, stainless steel or gold) are nowadays commercially available, the material of the target plays only a minor role under practical laboratory conditions: using dried droplet preparations, the sample/matrix layer is normally such thick that the laser (that normally penetrates only a few lm into the sample) irradiance does not depend on the composition of the target material. A more detailed discussion of aspects of sample preparations is available in [41] Typical MALDI matrices Fig. 3. Survey of some important UV MALDI matrices that are often used in the field of lipid analysis. The molecule classes from which the individual matrices are derived are also indicated. Please note that this is only a selection of the (in these authors opinion) most important matrices in the field of lipid analysis. Among the hundreds of compounds that were suggested to be useful as MALDI matrices, only a handful is used in daily practice and many suggestions of matrices remained a flash in the pan. As a complete survey of all matrix compounds (for a more detailed review see the rather old but still very useful paper by Fitzgerald [42]) would be beyond the scope of this paper, only the most important matrices in the lipid field will be discussed in more detail in the subsequent paragraphs of this review. A more complete survey is available in [29].

6 B. Fuchs et al. / Progress in Lipid Research 49 (2010) However, we would like to emphasize that lipids offer a considerable advantage in comparison to polar molecules: as already indicated above, the majority of MALDI devices make use of a UV laser emitting at 337 nm. This normally requires matrix compounds with aromatic residues. Such compounds are only barely soluble in water, but well soluble in organic solvents. Thus, spectra of apolar molecules can be recorded in a single organic phase without the need of adding water. This results in enhanced reproducibility and only relatively moderate shot-to-shot deviations [43] that are often serious problems regarding the MALDI MS analysis of polar molecules Inorganic MALDI matrices As the matrix is normally applied in vast excess over the analyte, typical matrix peaks (often representing cluster ions with higher masses than the original matrix compound) are normally seen in the MALDI spectra particularly in the smaller m/z range. This may lead to reduced sensitivities and the potential suppression of analyte signals. Therefore, the application of inorganic compounds that do not give any signals is a straightforward approach. The very first application of an inorganic matrix was published by Tanaka and coworkers [15] who used glycerol suspensions of cobalt nanoparticles for the ionization of large molecules. The performance of lm size metal and metal oxides such as Al, Mn, Mo, Si, Sn, SnO 2, TiO 2,W,WO 3, Zn and ZnO for the detection of small organic molecules was also tested by Kinumi [25] using polyethylene glycol 200 and methyl stearate as selected small analytes. Higher sensitivities in comparison to classical organic matrices could be obtained and no interfering matrix background signals were detected. Generally, decreasing particle size results in increased sensitivity and, thus, nanoparticles instead of microparticles are nowadays preferentially used. Although these metal or metal oxide particles do not give any matrix peak, they have a very high melting point and, thus, enhanced laser fluences have to be used to transfer them into the gas phase. This often results in enhanced analyte fragmentation [44]. Another problem that hinders their wider application is their limited commercial availability. Carbon nanotubes could be also successfully used and it has been shown that detection limits of certain analytes can be improved for one or two orders of magnitude [45]. An additional advantage of these materials is that they can be used to enrich certain apolar analytes due to their considerable hydrophobicity. Inorganic materials such as silicon [46], graphite [47] and TiO 2 [48] have been used to coat the surface of the MALDI plate, and no additional matrices were used. Fabrication of complete MALDI plates from porous silicon and graphite for matrix less analyte detection has also been reported [49]. Such inorganic oxides are especially useful because their isoelectric points vary over a wide range from acidic to basic. Therefore, the creation of positive or negative ions is favored depending on the oxide choice. Some selected applications of these matrices will be described at the appropriate places in this review. However, inorganic matrices are not commonly used so far and there are only very few applications to phospholipids available [50]. However, metal or metal oxide nanoparticles seem particularly promising in the imaging field because they give higher resolutions than standard solid matrices [51]. Therefore, all these materials are assumed to have significant future potential. be found in living organisms. As we are sure that the readers of Progress in Lipid Research will know the related chemical structures, we will pay only minor attention to this point. Our previous review in this journal [19] contained a survey of the related chemical aspects and may be consulted if needed. Additionally, there was a recent paper that gave a comprehensive survey of the classifications of lipids [53] Hydrocarbons and wax esters As already indicated above, positive ion MALDI MS is much more common than negative ion MALDI MS. In order to convert a (neutral) molecule into a positively-charged ion, a cation, for instance a proton must be added. Expectedly, this normally takes place at sites with a high electron density, i.e. charged groups such as phosphate or atoms with an electron lone pair such as nitrogen or oxygen. Nevertheless, even completely apolar compounds (without any oxygen, nitrogen or sulfur heteroatoms) such as the unsaturated hydrocarbon squalene (the structure of which is shown in Fig. 4), a hydrocarbon containing several isoprene units, as well as its oligomerization products can be easily analyzed by MALDI-TOF MS [54]. Squalene occurs in olive oil (about g/kg) and has been assumed to be related to the health benefits of vegetable oils and is, thus, of significant nutritional interest. Due to the apolarity of squalene, an auxiliary reagent such as silver trifluoroacetate (AgTFA) is often added to the matrix, for instance, DHB [55] in order to improve the yield of ions. As many hydrocarbons contain olefinic residues and give, thus, rise to a significant UV absorption, simple laser desorption (LD) MS can also be used because an additional matrix is not absolutely necessary and does not significantly improve the spectral quality. This provides the significant advantage that there is no interference with matrix peaks at all. However, direct LD analysis is impossible if completely saturated hydrocarbons are to be analyzed as these compounds lack sufficient UV absorptions. Asphaltene fractions of crude petroleum [56] can be investigated in a similar way and also in this case the MALDI and the LDI spectra are of comparable quality. Additionally, applications of MALDI-TOF MS to polyaromatic compounds [57] have also been described although the insolubilities of these molecules conferred some problems. These problems were solved by using a new sample preparation method consisting of mechanically mixing analyte and matrix without the necessity to use solvents [58]. Finally, 7,7,8,8-tetracyanoquinomethane (TCNQ) was shown to possess superior properties in comparison to other matrices [59]. A more comprehensive survey about LD MS is available in [60] and this reviews covers also aspects of desorption/ionization on silicon (DIOS) as well as carbon-based microstructures. Finally, a quite old, but nevertheless excellent review dealing with LD MS is available in [61]. 3. A survey of lipids so far investigated by MALDI-TOF MS Clearly, biopolymers and particularly proteins or peptides derived thereof are so far primarily investigated by MALDI-TOF MS [52]. In this review we will deal exclusively with apolar compounds and particularly with lipids and phospholipids that can Fig. 4. Chemical structure of the physiologically important hydrocarbon squalene. Virgin olive oil is a rich source of this hydrocarbon and the squalene content of vegetable oils is often assumed to be important for the related health benefits.

7 456 B. Fuchs et al. / Progress in Lipid Research 49 (2010) It should be noted that the lithium salt of DHB is a more suitable matrix for hydrocarbon analysis than the free acid DHB: LiDHB provides less pronounced fragmentation and higher sensitivity [62]. Similar data were also obtained in the case of wax esters [63] and insect cuticular hydrocarbons [64], whereby saturated and unsaturated hydrocarbons with more than 70 carbon atoms could be detected. In a very new paper it was shown that the distribution of different wax esters and other apolar compounds can also be investigated by MALDI imaging MS [65]. Of course, there are also many different apolar polymers the analysis of which might be of significant interest. These compounds will not be discussed here but the interested reader is referred to the excellent review by Batoy and coworkers [66] Plant pigments Since a pioneering MALDI work in 1996 [67], only very moderate efforts were undertaken to study chlorophylls and other plant pigments [68]. This is somewhat surprising because these pigments play a very central role in the plant metabolism, particularly regarding photosynthesis. Using standard DHB matrix, such molecules are easily detectable. However, they are only detectable subsequent to removal of the central metal ion [69]. Chlorophyll a, for instance, is detected at m/z = 871.5, although its monoisotopic mass is (for chemical structure see Fig. 5). The observed mass difference may be explained by the addition of three H + and the loss of Mg 2+ leaving a single positively-charged ion. A very recent study [70] provided evidence that the loss of the central ion can be avoided if terthiophene is used as the MALDI matrix. In contrast, however, fragmentation of the phytol-ester linkage was more pronounced in the presence of the terthiophene matrix. It seems likely that an enhanced laser fluence was needed under these conditions due to the weaker UV absorption of this matrix resulting in enhanced fragmentation Flavonoids and carotenoids These compounds can be easily characterized by MALDI-TOF MS although their tendency to give fragment ions seems strongly dependent on the applied matrix. According to current knowledge, Fig. 5. Chemical structure of chlorophyll, a very important plant constituent that enables light fixation during the photosynthesis process. only a minor extent of fragmentation is detectable if 2 0,4 0,6 0 -trihydroxyacetophenone (THA) is used as matrix, whereas DHB gives much higher yields of fragment ions and is, thus, the matrix of choice to record post source decay (PSD) mass spectra [71]. It was also reported in the context of red wine analysis that quantitative data can be directly achieved from the MALDI mass spectra [72]. Due to the absorbance of these compounds in the UV range, recording simple laser desorption spectra is a potential alternative. It has been recently shown that direct imaging of plants is also possible [73]: as no matrix addition is required (that determines the achievable resolution due to the size of the crystals), highly resolved (resolution about 10 lm) MS images can be obtained and it could be shown that the highly specific distribution of important flavonoids such as kaempferol, quercetin and isorhamnetin can be imaged at the cellular level. Similar data can be also obtained if colloidal graphite is used as matrix [74]. In a very recent work it could be shown that flavonoids such as quercetin or rutin may be used themselves as matrices for inorganic metal complexes [75] and provide better results in comparison to common crystalline matrices such as DHB Free fatty acids The MALDI-TOF MS analysis of free fatty acids is quite difficult if standard MALDI matrices are used. The most serious problem is the overlap of the signals of the free fatty acids with matrix signals that are particularly abundant in the low mass range. Of course, this is a serious problem if fatty acids at low concentrations have to be analyzed. According to our best knowledge standard matrices such as DHB or CHCA (a-cyano-4-hydroxycinnamic acid) are not suitable for this purpose, but some methods as to how this problem can be overcome have been suggested: 1. If meso-tetrakis(pentafluorophenyl)porphyrin (MTPFPP), the structure of which is shown in Fig. 6 [76], is used as matrix, the problem of signal and matrix overlap can be overcome. MTPFPP has a relatively high mass and does not give signals below m/z ffi 500 in the positive-ion mode. Thus, fatty acids with typical masses between about 200 and 350 Da can be unequivocally detected. This was demonstrated, for instance, with fatty acid mixtures obtained from different vegetable oils subsequent to alkaline hydrolysis [77]. As an excess of sodium acetate was added, exclusively the Na + adducts of the Na + salts of the released fatty acids were detected. Therefore, there are no problems with the overlap of different adducts, on the one hand, and differences in fatty acyl compositions (the H + adduct of arachidonic acid (20:4) results in the same m/z ratio as the Na + adduct of oleic acid (18:1)), on the other hand, could be completely avoided. This method was also successfully used for the analysis of complex fatty acid mixtures from different biological samples, for instance, rat plasma [78]. However, MTPFPP is so far not widely used because this matrix gives rise to unknown artifacts. Typical examples of positive ion mass spectra of selected free (saturated (6a) as well as unsaturated (6b)) fatty acids recorded in the presence of MTPFPP are shown in Fig. 6. Although all saturated fatty acids are detected with the expected m/z ratios, a mass shift of 14 amu to higher masses is observed if unsaturated fatty acids are investigated [79]. For instance, oleic acid is observed at m/z = (Na + adduct of the sodium salt) as well as at m/z = Although this effect has not been carefully investigated yet, the above data might indicate the oxidation of a methylene into a carbonyl group. Additionally, the sensitivity of this MTPFPP matrix is rather poor and at least lg quantities of fatty acids are required. 2. Fatty acids are acidic compounds and should be, thus, easily detectable as negative ions at least if a sufficiently

8 B. Fuchs et al. / Progress in Lipid Research 49 (2010) alkaline matrix is used. 9-Aminoacridine (9-AA) seems a particularly promising matrix in this field due to its considerable basicity (the pk a is about 9.99) [80]. Another advantage of 9-AA is the very moderate background [81] provided by this matrix. It could be shown that the achievable sensitivity is in the femtomolar range and linear detector response could be obtained over two orders of magnitude. Therefore, 9-AA seems the matrix of choice for quantitative metabolomics studies of negatively charged compounds not only fatty acids [82]. 3. Very recently [83], it could be shown that 1,8-bis(dimethylamino)naphthalene (DMAN), a superbasic compound with a pk a of [84] is a powerful matrix because it enables the detection of fatty acids (saturated and unsaturated ones) in low picomole amounts. Therefore, both, 9-AA and DMAN seem more suitable than MTPFPP because all fatty acids can be easily and accurately detected, while the MTPFPP porphyrin matrix is obviously less suitable for unsaturated fatty acids (cf. Fig. 6b) due to the +14 amu artifact and the lower achievable sensitivity. However, there are rumors that the use of DMAN may decrease the sensitivity in the positive-ion mode significantly: residual amounts of DMAN may bind all available protons and reduce the achievable sensitivities by reducing the available amount of this cationizing species. 4. Free fatty acids are also detectable if inorganic matrices such as graphite [85] or porous silicon [86] (desorption/ionization on porous silicon (DIOS MS)) are used. Although the achievable sensitivity is rather poor, deprotonated fatty acids could be easily detected as negative ions. A more detailed survey of this topic is available in [87] Cholesterol and cholesteryl esters Although cholesterol is present in virtually all mammalian cells and body fluids [43] as well as in combination with significant amounts of cholesteryl esters (CE) and triacylglycerols (TAG) in the lipoproteins of blood, only little interest has been paid to the MALDI MS characterization of these molecules. One potential reason is the commercial availability of enzymatic test kits that help to determine both, cholesterol and cholesteryl ester concentrations making MS methodology less important for the determination of these molecules. Using established MALDI matrices such as DHB, cholesterol is not detectable as the expected H + adduct but only subsequent to water elimination at m/z = (M+H + H 2 O) [88]. Although it has been shown that the cholesterol concentration in extracts of, e.g. human lipoproteins can be accurately determined by MALDI-TOF MS [89], the relatively small mass of cholesterol confers problems: in the same manner as in the case of free fatty acids, there is a considerable overlap between the cholesterol peak and the matrix background. Of course, this is a particularly important problem if diluted samples have to be analyzed. To our best knowledge, there were so far no attempts to establish a more suitable matrix for cholesterol analysis. In particular it has not yet been clarified, whether 9-AA represents a useful alternative matrix: 9-AA normally detects only charged lipids such as phospholipids, while an additional cationizing reagent (e.g. sodium acetate) is required to generate ions from molecules that do not possess charged functional groups. These aspects will be discussed below in more detail and it will be outlined that the combination of different matrices represents the method of choice to obtain a complete data set [90]. Fig. 6. Positive ion MALDI-TOF mass spectra of mixtures of fatty acids using MTPFPP as matrix. In (a) a mixture of lauric (12:0), myristic (14:0) and palmitic acid (16:0) was investigated, whereas in (b) a mixture of oleic (18:1), linoleic (18:2) and a-linolenic acid (18:3) was applied. Reprinted with permission and with slight modification from Journal of Food Lipids, 9 (2002) [79].

9 458 B. Fuchs et al. / Progress in Lipid Research 49 (2010) There have been surprisingly few attempts to investigate cholesteryl esters by MALDI MS [89,91]: in the same way as observed for di- and triacylglycerols (see below), CEs are exclusively detectable as adducts with alkali metal ions (particularly sodium), whereas the H + adducts are not detectable at all. Although not yet carefully investigated, this might be caused by the same reason as described for triacylglycerols [92]: H + adducts of cholesteryl esters are less stable in comparison to the Na + adducts and do not survive the flight distance to the mass analyzer of the MALDI device without fragmentation. The experimental fact that the MALDI mass spectra of chromatographically pure CEs always exhibit a small cholesterol peak is a strong confirmation of this mechanism Sphingolipids Sphingolipids and glycosphingolipids are currently a hot research topic because these compounds may be regarded to be indicative of ageing and as disease markers. As there are some recent reviews dealing with the subject of sphingolipidomics [93,94], we will deal here only rather shortly with these compounds but will discuss this topic in more detail in the context of TLC/MALDI (see below). Here, we will focus primarily on the analysis of the most abundant sphingolipid, sphingomyelin (SM), the structure of which is shown in Fig. 7. SM is detectable in the same way as phospholipids and H + as well as Na + adducts occur. Very recently a combination between MALDI and ESI MS was used to study changes of the sphingolipid composition of parasitic nematodes [95]. It could also be proven that changes of the lipid composition of tissues (and particularly of brain) often affect much more the sphingolipids than the glycerophospholipids [96]. Additionally, SM is an important constituent of virtually all body fluids as well as tissues and, thus, particularly important in the context of imaging studies of tissues [97]. Surprisingly, in comparison to our previous review [19], only a few papers dealing with the MALDI MS analysis of sphingolipids appeared. Nevertheless, it became obvious that a large variety of sphingolipids can be quantitatively analyzed by MALDI-TOF MS in the presence of an internal standard [98]: with sphingosylphosphorylcholine as the internal standard, the relative peak heights of SM and ceramide monohexoside (CMH) could be used as a quantitative concentration measure and linearity could be obtained between 50 and 1500 ng SM as well as 5 and 150 ng CMH content, respectively. Nevertheless, despite the similarities of the headgroups, SM is less sensitively detectable in comparison to PC Glycerolipids Triacylglycerols (where all hydroxyl groups of the glycerol are esterified with fatty acids) are very abundant in animal organisms, whereas compounds such as mono- or digalactosylglycerols are particularly abundant in plants [99]. We will focus here primarily on animals and only to a lesser extent on plant lipids. Therefore, characteristics of TAGs (and to a minor extent diacylglycerols) will be primarily discussed. Fig. 7. Chemical structure of the backbone of sphingomyelin. R represents a variable alkyl chain Di- and triacylglycerols Triacylglycerols (TAG) are important for the storage of energy (fat tissues in living organisms), while diacylglycerols (DAG) that are normally generated from glycerophospholipids by cleavage of the polar headgroup under the influence of the enzyme phospholipase C are important lipid-derived second messengers [100]. The positive ion MALDI-TOF mass spectra of both, DAGs [101] as well as TAGs [102] can be easily recorded with standard DHB. The MALDI mass spectra give always exclusively the Na + adducts, whereas H + adducts are never detected, even if the solutions are acidified [92]. Additionally, there are always intense fragment ions corresponding to the loss of one sodiated fatty acyl residue. Using MALDI MS in combination with high energy collisionally-induced dissociation (CID), the losses of these fatty acyl residues can be used for structural elucidation of TAGs [103], i.e. the determination which fatty acyl residue is located in which position. It is important to note that exclusively the Na + adducts were useful for that purpose while neither the K + nor the Li + adducts (that can be easily obtained if the spectra are recorded in the presence of the corresponding alkali salts) gave indicative fragment ion spectra [103]. The applied matrix has only a relatively weak impact on the spectral quality, whereas strongly different sensitivities are achieved in dependence on the used solvents. For instance, CHCA and DHB dissolved in a mixture of acetonitrile and water gave only sensitivities in the pmol range, whereas DHB in acetone provided detection limits in the fmol range. Additionally, DHB provides a weaker background than cinnamic acid derivates [104] that tend to significant matrix cluster generation. This is even more important than the achievable sensitivities because TAGs (e.g. from vegetable oils) are normally available in huge amounts. In order to have highly reproducible spectra, sample preparation is very important and particularly the ion content must be carefully controlled. For instance, it was recently shown that 9-AA detects TAG only in the presence but not in the absence of additional cationizing reagents ( dopants ) such as ammonium acetate [105]. It is somewhat surprising that independent of the used matrix, DAG and TAG appear exclusively as the sodiated molecules, whereas the H + adducts are not detectable at all. A convincing explanation has been recently given [92] and is based on the observation that the generation of fragment ions can be significantly reduced under alkaline conditions. Thus, it was suggested that the observed fragments actually arise from unseen protonated TAGs as their fragmentation occurs so rapidly and completely that protonated TAGs are normally not observed. If the ph is increased, the H + concentration is simultaneously decreased leading to reduced H + adduct generation and, accordingly, to a lower yield of fragmentation products [92]. Very recently it has been shown that the yield of TAG fragments can be significantly reduced if the MAL- DI target is covered with a thin nitrocellulose film prior to deposition of the TAG of interest [106]. The nitrocellulose film simultaneously improves the shot-to-shot and sample-to-sample reproducibility through the exhibition of a more homogeneous matrix/analyte co-crystallization. It has been repeatedly shown that MALDI MS is very suitable for the screening of the compositions of crude TAG mixtures, for instance vegetable oils such as olive oil [107], cod liver oil [108] and animal fat such as milk fat, butter, beef tallow or lard [109]. Due to the large structural variabilities of TAG in different samples, methods of bioinformatics (such as principal component analysis) play an increasingly important role regarding data analysis [110]. In order to clarify the structures of TAG species that could not be unequivocally assigned, bromination and hydrogenation was also applied and combined with PSD fragment ion spectra [109]. Another important application of MALDI-TOF MS is the investigation of the changes occurring in oil samples during frying [111]. This is a very important practical application because products