progress in lipid research - maldi tofmaldi.ch.pw.edu.pl/pomiary/artykuly/an update of maldi-tof...

Progress in Lipid Research 49 (2010) 450–475

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Progress in Lipid Research

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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

a r t i c l e i n f o a b s t r a c t

Article history:Received 25 April 2010Received in revised form 29 June 2010Accepted 1 July 2010

Keywords:MALDI-TOF MSLipidsPhospholipidsMatrix

0163-7827/$ - see front matter � 2010 Elsevier Ltd. Adoi:10.1016/j.plipres.2010.07.001

Abbreviations: 9-AA, 9-aminoacridine; amu, atomaacid; CID, collisionally-induced dissociation; DAG, ddihydroxybenzoic acid; DIOS, desorption/ionizationaluminium garnet; ESI, electrospray ionization; FAB, fdesorption/ionization; GC, gas chromatography; GPL,IP, ionization potential; IR, infrared; LD, laser desphosphatidylcholine; LPL, lyso(monoacyl)-phospholispectrometry; MTPFPP, meso-tetrakis(pentafluoropheresonance; PA, phosphatidic acid; PC, phosphatidylch4,5-bisphosphate; PL, phospholipid; PLA2, phospholippolyvinylidene difluoride; RF, retardation factor; ROSsecondary ion MS; SM, sphingomyelin; S/N, signal to3-methoxycinnamic acid; TFA, trifluoroacetic acid; TH

* Corresponding author. Tel.: +49 341 97 15733; faE-mail address: [email protected]

Although matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) – often butnot exclusively coupled with a time-of-flight (TOF) mass analyzer – is primarily established in the proteinfield, 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 spectrawithout 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 spec-trometric characteristics of the individual lipid (ranging from completely apolar hydrocarbons to com-plex glycolipids with the focus on glycerophospholipids) classes will be discussed and the progressachieved in the last years emphasized. Special attention will be paid to quantitative aspects of MALDIMS because this is normally considered to be the ‘‘weak” point of the method, particularly if complexlipid 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 detectall compounds of interest.

Two rather recent developments will be highlighted: ‘‘Imaging” MS is nowadays widely establishedand significant interest is paid in this context to the analysis of lipids because lipids ionize particularlywell 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 highersensitivity than common staining protocols.

� 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
1.1. Soft ionization mass spectrometric methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4511.2. Fundamentals of MALDI mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
2. The role of the matrix – some practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
2.1. Typical MALDI matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4542.2. Inorganic MALDI matrices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
ll rights reserved.

r mass unit; APCI, atmospheric pressure chemical ionization; CE, cholesteryl ester; CHCA, a-cyano-4-hydroxycinnamiciacylglycerols; DE, delayed extraction; DESI, desorption electrospray; DGDG, digalactosyl-diacylglycerol; DHB, 2,5-on silicon; DMAN, 1,8-bis-(dimethylamino)-naphthalene; EI, electron ionization; Er:YAG, erbium-doped yttrium

ast atom bombardment; FD, field desorption; FI, Field Ionization; FT, Fourier Transform; GALDI, graphite-assisted laserglycerophospholipid; HPLC, high-performance liquid chromatography; HPA, hydroxy-picolinic acid; ILS, ionic liquids;orption; LOD, level of detection; LOQ, level of quantification; LPA, lysophosphatidic acid; LPC, lyso(monoacyl)-pid; MALDI, matrix-assisted laser desorption and ionization; MGDG, monogalactosyl-diacylglycerol; MS, massnyl)porphyrin; m/z, mass over charge; Nd:YAG, neodymium-doped yttrium aluminium garnet; NMR, nuclear magneticoline; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP2, phosphatidylinositol-ase A2; PNA, para-nitroaniline; PPI, (poly-)phosphoinositides; PS, phosphatidylserine; PSD, post source decay; PVDF,, reactive oxygen species; S1P, sphingosine-1-phosphate; SA, sinapic acid; SCX, silica gel cation exchanger; SIMS,

noise; sn, stereospecific numbering; SQDG, sulfoquinovosyl-diacylglycerol; TAG, triacylglycerol; TCA, trans-4-hydroxy-A, trihydroxy-acetophenone; TLC, thin-layer chromatography; TOF, time-of-flight; UV, ultraviolet.

x: +49 341 97 15709.ig.de (J. Schiller).

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B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 451

3. A survey of lipids so far investigated by MALDI-TOF MS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3.1. Hydrocarbons and wax esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4553.2. Plant pigments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4563.3. Flavonoids and carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4563.4. Free fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4563.5. Cholesterol and cholesteryl esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4573.6. Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4583.7. Glycerolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.7.1. Di- and triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4583.7.2. Glycoglycerolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459

3.8. Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.8.1. Zwitterionic glycerophospholipids (PC and PE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4593.8.2. Acidic glycerophospholipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
4. Analysis of lipid mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4645. Separation of the individual lipid classes prior to analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466

5.1. Glyco- and sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4675.2. Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468

6. MALDI MS imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4697. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

1. Introduction

We are currently living in an ‘‘omics” time period [1]. Althoughthis list is surely not complete, there were already proteomics,genomics, lipidomics, metabonomics, metagradomics, metallomicsand – rather recently – interactomics. As these approaches providean immense amount of data, bioinformatics is an indispensablecounterpart leading to ‘‘systems biology” initiatives [2] that mayhelp to get further insights into complex metabolic networks ofbiological systems.

As this review is dedicated to lipids and their analysis, a shortdefinition of ‘‘lipidomics” is necessary: according to a recently pro-vided definition [3], ‘‘lipidomics” can be defined as ‘‘the full charac-terization of lipid molecular species and of their biological roleswith respect to expression of proteins involved in lipid metabolismand function, including gene regulation”. The combination of allthese complex aspects is clearly a very challenging task, but thefirst step is obviously the qualitative and quantitative knowledgeof the lipid composition of an unknown sample.

Many ‘‘omics” applications are based on the application of massspectrometry (MS) and would not have been possible without thesignificant progress achieved in this field during the last decades.MS is an incredible powerful method and may be regarded as anindispensable 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 ini-tially related to the discovery of the different isotopes of the chem-ical elements [5] and, thus, primarily a task of physicists. Forinstance, Thompson was awarded the Nobel Prize (of Physics) in1906 for his work on ‘‘Conduction of Electricity through Gases”.

In contrast, the history of the success of MS in biology and lifesciences is much shorter and directly related to the discovery ofnew and gentle ionization methods: over many decades electronionization (EI) was exclusively available as ionization method [4].EI makes use of fast electrons to ionize the analyte molecules inthe gas phase by the removal of one electron. Although this tech-nique was (and still is) unequivocally suitable for the investigationof small and/or volatile compounds, EI normally fails if larger mol-ecules with low volatilities are to be analyzed: although it could beshown that the molecular ion (M ? M�+ + e�) of phospholipids isbasically detectable if EI is used for ionization of the sample, theobtained results were not very convincing [6] and fragment ionsare much more abundant in comparison to M�+ making this tech-

nique less suitable for the analysis of mixtures. Therefore, theprime application of EI MS in the context of lipids is nowadays nor-mally the analysis of free fatty acids that can be obtained by sapon-ification of lipids. This also holds for oxidation products derivedfrom free fatty acids and their metabolic products such as throm-boxanes or leukotrienes [7]. GC/MS is even nowadays a highlyestablished and widely used method for the quantitative analysisof relatively apolar compounds. Unfortunately, sample preparationis quite cumbersome and time-consuming. Because of these disad-vantages, the invention of soft-ionization methods, enabling theanalysis of molecules refractive to EI, must be regarded as a realmilestone in the history of modern mass spectrometry [8].

1.1. Soft ionization mass spectrometric methods

The differentiation between ‘‘hard” and ‘‘soft” ionization MSmethods is a matter of philosophy and depends to a significant ex-tent on the analyte of interest and its properties. However, all mod-ern ionization methods may be regarded as ‘‘softer” than EI [9].Methods enabling the ionization of compounds refractive to elec-tron collision ionization comprise gas phase ionization techniquessuch as chemical ionization (CI) that was discovered in 1913 byThompson [10], field desorption (FD) and field ionization (FI),methods based on particle bombardment such as fast atom bom-bardment (FAB) or secondary ion MS (SIMS) [11]. Each of these ion-ization methods has its individual strengths and weaknesses, butthe application of these methods is normally limited to specialproblems or selected analytes [4]. Only chemical ionization (oftenin combination with atmospheric pressure, APCI) is used to a high-er extent for the analysis of lipids. For a recent review of applica-tions of CI in lipid research see [12], while a survey of theadvantages and drawbacks of the different ionization techniquesis available in [13].

Today, electrospray ionization (ESI) and matrix-assisted laserdesorption and ionization (MALDI) MS are most often used andthe majority of commercially available MS devices is equippedwith one (or maybe even both) of these ion sources. As a maximumof information is obviously achievable if both techniques are com-bined, they should be regarded as complementary but not as com-petitive. The importance of these both ionization methods wasobviously 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].

452 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

We will not provide here a comprehensive survey of the funda-mentals of ESI MS (for reviews dedicated to ESI MS analysis of lip-ids see [17,18]), but will focus on applications of MALDI MS and therelated methodology. A review with a similar topic was alreadypublished by our group in 2004 in ‘‘Progress in Lipid Research”[19]. However, many important improvements regarding potentialapplications, matrix optimization, quantitative data analysis andmore detailed characterization (particularly by MS/MS) of thedifferent lipid classes have been achieved since that time and anupdate of our previous review seemed necessary. In our opinionthe most important developments can be summarized as follows:

1. Although details of ion generation are still not yet completelyunderstood, some progress in understanding this importantaspect has been achieved. This is very important because fur-ther theoretical insights will help to improve the performanceof the MALDI method. A comprehensive survey of theoreticalaspects is available in [20].

2. Some new and improved matrix compounds were introducedthat provide either higher sensitivities or higher reproducibili-ties than previously used matrices [21]. We will emphasize herethat some lipid-derived compounds are exclusively detectable ifthe most appropriate matrix is used.

3. The coupling between MALDI MS and chromatographic separa-tion techniques is unequivocally a ‘‘hot” topic of currentresearch. This particularly concerns the combination betweenMALDI MS and thin-layer chromatography (TLC) that helps toovercome many problems related to mixture analysis in a sim-ple but elegant way [22].

4. MS imaging is nowadays an established method to monitor thedistribution of certain molecules within a tissue. As lipids ionizeparticularly well, they are very easily detectable under theseconditions and this fact has pushed the interest in lipid analysisby MALDI MS considerably [23].

The continuously increasing interest in MALDI MS as well as itsapplications in lipid analysis is clearly reflected by the data given inFig. 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 lastcenturies there were a lot of ‘‘omics” initiatives and many MALDIdevices were purchased – particularly for protein and peptideanalysis. It is our aim to convince the reader that these instrumentsare not exclusively useful in the proteomics field, but the sameinstruments can be readily used for the analysis of lipids. Thereis surely no need to buy an additional device but the availableMALDI devices can be used without the need of major alterations!

Fig. 1. Number of scientific papers containing the term ‘‘MALDI” or ‘‘matrix-assisted” (a)were taken from the ‘‘Web of Science”™ database.

1.2. Fundamentals of MALDI mass spectrometry

A detailed survey of methodological aspects of MALDI MS isavailable in the excellent book by Franz Hillenkamp and JasnaPeter-Katalinic [20]. Therefore, theoretical fundamentals of MALDIMS will be only shortly discussed in this review. However, a shortintroduction is necessary for the less experienced reader.

MALDI MS is based on the utilization of a ‘‘matrix” that initiallyabsorbs the energy of the laser and mediates the generation of ions.Although inorganic compounds such as graphite [24] or metaloxide particles [25] may be also applied as matrix, small organicmolecules are used in the majority of cases and will be, thus, nearlyexclusively discussed here.

Of course, the suitability of a certain compound as matrix isdetermined by the type of the laser and its emission wavelength.Although many different lasers, including Nd:YAG (k = 355 or266 nm) [26], excimer [27] or CO2 lasers [28] were already success-fully applied, in the lipid field N2 lasers are primarily used.Although IR lasers provide some advantages (see below), we willfocus here nearly exclusively on UV lasers with an emission wave-length of 337 nm because the majority of commercially availableMALDI devices are equipped with this laser type. This is the reasonwhy the majority of the so far available lipid data were obtainedwith UV lasers. A more comprehensive discussion of the role ofthe matrix is available in [29].

When the pulsed laser beam hits the sample (normally co-crystals of the matrix and the analyte), its energy is primarily ab-sorbed by the matrix that is present in a vast excess over the analyte(a 100–100,000-fold excess of the matrix is typically used). Conse-quently, the matrix is vaporized, carrying intact analyte moleculesinto the vapor phase. A simplified schema of the processes occurringin a typical MALDI-TOF mass spectrometer is shown in Fig. 2 [19].

During the expanding process of this gas cloud, ions (e.g. H+ andNa+) are exchanged between the matrix and the analyte, leading tothe formation of charged analyte molecules. These analyte ions arecalled ‘‘adducts” or ‘‘quasimolecular ions” (sometimes the term‘‘pseudomolecular” ions may be also found although the use of thisterm is discouraged by the IUPAC). Beside cation generation, an-ions can also be generated by abstracting H+ or Na+ from the ana-lyte. The ratio between the cation and the anion yield isdetermined by the (gas phase) acidities of the analyte and the ma-trix [20] and fundamentals of the ion formation process were re-cently comprehensively reviewed by Zenobi and Kochenmuss[30,31]. Recording positive-ion mode spectra is much more com-mon and it even seems (although not yet investigated in detail)that many MALDI mass spectrometers detect negative ions lesssensitively than positive ions.

and papers that contain additionally the term ‘‘lipid” or ‘‘phospholipid” (b). All data


The most important difference between ‘‘molecular (radical)ions” (generated in conventional EI mass spectra) and ‘‘quasimo-lecular ions” is the observed mass. As molecular ions are generatedby the abstraction of one electron (the mass of which can be ne-glected for the majority of applications because it accounts for justabout two-tenths of a percent of the mass of a proton) from theanalyte, the mass of the generated ion (or the observed m/z ratio)corresponds to the mass of the analyte. In contrast, quasimolecularcations are generated by the addition of a cation to the analyte.Therefore, the mass of the quasimolecular ion is characteristicallyhigher (normally 1 amu or 23 amu corresponding to the massof H+ or Na+, respectively) in comparison to the analyte molecule[32].

Despite its profound importance, the process of ion generationis so far only poorly understood and many papers are currentlydealing with this important topic. For instance, one recent issueof European Journal of Mass Spectrometry (2006, Volume 12, Issue6) was exclusively dedicated to the topic ‘‘Mechanisms of MALDI”.Despite this obvious lack of knowledge, it is sure that singly-charged ions are primarily generated [33] and, therefore, the actualmeasured quantity, the mass-to-charge ratio (m/z) may be re-placed 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 ratiowas suggested nearly 20 years ago [34] but is not yet widely estab-lished. A detailed discussion of this aspect is available in [35].

After being formed, ions are accelerated in an electric field (typ-ically of the order of 20 kV). After passing a charged grid, the ions aredrifting freely over a field-free space where mass separation isachieved: low mass ions arrive at the detector in a shorter time thanhigh mass ions [13]. This is the most simple, ‘‘linear” geometry of aTOF analyzer that is normally used for the analysis of larger mole-cules because in this case high resolution is less important than highsensitivity. Resolution and peak widths may be improved by using areflectron [20]. The reflectron enlarges the flight path and helps tocompensate differences in the initial velocities of the ions duringthe ablation process. All spectra shown in this review were recordedon a MALDI-TOF device equipped with a reflectron. Using commer-cial MALDI-TOF devices with reflectron configuration, mass resolu-tions of about 10,000 and mass accuracies higher than about 30 ppmcan be routinely achieved. This is rather poor in comparison to thebest available mass spectrometers nowadays which provide massaccuracies lower than 1 ppm and also much higher resolutions. Suchhigh quality mass spectra are desirable in the context of proteomics,where an increased mass accuracy helps to unequivocally assign un-known (tryptic) peptides because the number of hits upon databasesearching is significantly reduced [36] if the mass can be providedwith higher accuracy.

Sample

Matrix

H

Nitrogen Laser(337 nm)

High Voltage

SamplePlate

ChargedGrid

FTarget

Fig. 2. Schema of the processes occurring during the MALDI-TOF ionization process in th‘‘linear” and ‘‘reflector” mode is emphasized in the figure. Reprinted with modification

In the context of lipids, however, very high mass accuracies donot really provide a much larger extent of information because thesmallest mass difference within one given lipid class is 2 amu, i.e.one double bond. We will show below that there are some simplechemical/biochemical methods (addition of certain salts or enzy-matic digest) to overcome problems related to potential signaloverlap and a MALDI device with a high resolving power is notabsolutely needed for routine lipid analysis.

It should be noted that there is no absolute need to combine aMALDI ion source with a ‘‘TOF” mass analyzer. However, as MALDIis used in the majority of cases for the detection of rather largemolecules, the TOF detector is very popular because it has a nearlyunlimited mass range [20]. An additional reason is the pulsed (notcontinuous) ion generation of MALDI that is most suitable for theTOF mass analyzer. A more comprehensive survey of typical massanalyzers as well as their individual advantages and drawbacks isavailable in [13,20].

2. The role of the matrix – some practical considerations

Although there were already different attempts to predict thesuitability of an unknown chemical compound as MALDI (UV) ma-trix by theoretical considerations, most matrices were (and actu-ally are) found by accident or by a ‘‘try and error” approach. Amore detailed discussion of these aspects is beyond the scope ofthis review and we will focus exclusively on some selected practi-cal aspects. A ‘‘good” MALDI UV matrix in the practical sense ischaracterized 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 sen-sitivity should be achievable.

2. The matrix should provide a high absorbance at the emissionwavelength of the laser. Thus, the required laser fluence shouldbe as low as possible because enhanced laser fluence leads toenhanced analyte fragmentation and/or poorly resolved massspectra.

3. An optimum matrix is characterized by low background (i.e. thesignals of the matrix should be very small) in order to avoidinterferences between the matrix and the analyte ions. It isimportant to note that oligomers of the matrix are often gener-ated in the gas phase. Therefore, most matrices provide signalsat m/z ratios much higher than their actual molecular weight!

4. Finally, an ideal matrix should provide only a single adduct ofthe analyte and exhibit a weak tendency to cluster formationbecause analyte–matrix clusters may seriously complicate dataanalysis [37].

Reflector("electrostatic Mirror")

eavy IonsLi ght Ionsmass to charge

(m/z)ield-free time-of-flight

LinearDetector

ReflectorDetector

mass to charge(m/z)

e mass spectrometer (for details see text). The influence of the detection using theand permission from Elsevier [19].


As the vast majority of commercially available MALDI devicesare equipped with nitrogen lasers, the focus of this review willbe on matrices compatible with N2 lasers (337 nm), whereas infra-red lasers and the corresponding matrices (such as glycerol orsuccinic acid) will be treated only briefly. There are basically fivetypical requirements that must be met by a powerful MALDImatrix and some selected UV MALDI matrix compounds are shownin Fig. 3.

(a) The matrix must exhibit a strong absorption at the laseremission wavelength, i.e. in the case of a UV laser normallyat 337 nm. This is the reason why nearly all common organicmatrices contain an aromatic ring system with delocalized pelectrons. Inorganic matrices such as graphite or metal saltswill only be shortly discussed here because they are scarcelyapplied to the field of lipid analysis so far [38]. As expected,the ionization efficiency increases if the absorption coeffi-cient of the matrix at the laser wavelength increases and thisis one reason why among the different isomers of dihy-droxybenzoic acids (DHB), only the 2,5 isomer that providesthe most marked absorption at 337 nm (but not the otherisomers) is commonly used as MALDI matrix [39]. Althoughthe solution UV absorption of the matrix is determined inmany studies, it must be emphasized that exclusively theUV absorption in the solid state determines the absorptionproperties under MALDI conditions. Absorptions determinedin the liquid and in the solid state may be significantly dif-ferent [39].

(b) The energy absorption and the subsequent evaporation ofthe matrix must lead to the generation of ions from the ana-lyte of interest. This is often believed to be the reason for theuse of carboxylic acids (many matrices are derived from cin-namic or benzoic acids) as matrix compounds because

Fig. 3. Survey of some important UV MALDI matrices that are often used in the fieldof lipid analysis. The molecule classes from which the individual matrices arederived are also indicated. Please note that this is only a selection of the (in theseauthors opinion) most important matrices in the field of lipid analysis.

organic acids are acidic and, thus, capable of triggering thegeneration of H+ adducts. As sodium is an ubiquitous ele-ment, however, there are normally also Na+ adducts in addi-tion to H+ adducts. In addition to the enhanced generation ofH+ adducts, there is another practical reason to use carbox-ylic 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 mole-cules that are only scarcely soluble in organic solvents, wateris one important constituent of the most common solventsystems: the presence of polar groups (such as carboxylicacids) 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 use-ful MALDI matrices that do not – or not sufficiently – fulfillthis criterion. As most MALDI devices make use of high vac-uum conditions (normally about 1 � 10�9 bar) many com-pounds tend to sublime. The loss of the matrix under highvacuum conditions may be one important reason whyMALDI mass spectra show time-dependent changes [40].

(d) The matrix should isolate the generated ions and prevent thegeneration of analyte clusters, for instance, dimer formation.This is the prime reason why a significant excess of thematrix over the analyte is normally required in order toobtain optimum results. Additionally, the laser fluenceshould be kept as high as needed but as low as possiblebecause matrix analyte clusters are increasingly generatedat elevated laser fluences [19].

(e) The matrix and the analyte should give homogenous co-crystals. Improvement of homogeneity is clearly science ofits own and depends – beside the analyte, the matrix andthe solvent system – on the method of sample preparation.This important topic would be a review of its own and hasbeen reviewed elsewhere [41] but with the focus on polarmolecules. Briefly, the simpler the sample preparationmethod, the lower is the homogeneity of the matrix/analytemixture. The most frequently used ‘‘dried droplet” method,i.e. the successive deposition of matrix and analyte are nor-mally not suitable if any quantitative data have to be derivedfrom the mass spectra. In contrast, the more sophisticatedsample preparation by electrospray deposition provides amuch more homogeneous matrix/analyte mixture and nor-mally permits quantitative data evaluation. Although MALDItargets made from different metals (e.g. aluminum, stainlesssteel or gold) are nowadays commercially available, thematerial of the target plays only a minor role under practicallaboratory conditions: using dried droplet preparations, thesample/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 targetmaterial. A more detailed discussion of aspects of samplepreparations is available in [41].

2.1. Typical MALDI matrices

Among the hundreds of compounds that were suggested to beuseful as MALDI matrices, only a handful is used in daily practiceand many suggestions of matrices remained ‘‘a flash in the pan”.As a complete survey of all matrix compounds (for a more detailedreview see the rather old but still very useful paper by Fitzgerald[42]) would be beyond the scope of this paper, only the mostimportant matrices in the lipid field will be discussed in more de-tail in the subsequent paragraphs of this review. A more completesurvey is available in [29].

Fig. 4. Chemical structure of the physiologically important hydrocarbon ‘‘squa-lene”. Virgin olive oil is a rich source of this hydrocarbon and the squalene contentof vegetable oils is often assumed to be important for the related health benefits.


However, we would like to emphasize that lipids offer a consid-erable advantage in comparison to polar molecules: as alreadyindicated above, the majority of MALDI devices make use of a UVlaser emitting at 337 nm. This normally requires matrix com-pounds with aromatic residues. Such compounds are only barelysoluble in water, but well soluble in organic solvents. Thus, spectraof apolar molecules can be recorded in a single organic phase with-out the need of adding water. This results in enhanced reproduc-ibility and only relatively moderate shot-to-shot deviations [43]that are often serious problems regarding the MALDI MS analysisof polar molecules.

2.2. Inorganic MALDI matrices

As the matrix is normally applied in vast excess over the ana-lyte, typical matrix peaks (often representing cluster ions withhigher masses than the original matrix compound) are normallyseen in the MALDI spectra – particularly in the smaller m/z range.This may lead to reduced sensitivities and the potential suppres-sion of analyte signals. Therefore, the application of inorganic com-pounds that do not give any signals is a straightforward approach.

The very first application of an inorganic matrix was publishedby Tanaka and coworkers [15] who used glycerol suspensions ofcobalt nanoparticles for the ionization of large molecules. The per-formance of lm size metal and metal oxides such as Al, Mn, Mo, Si,Sn, SnO2, TiO2, W, WO3, Zn and ZnO for the detection of small or-ganic molecules was also tested by Kinumi [25] using polyethyleneglycol 200 and methyl stearate as selected ‘‘small” analytes. Highersensitivities in comparison to classical organic matrices could beobtained and no interfering matrix background signals were de-tected. Generally, decreasing particle size results in increased sen-sitivity and, thus, nanoparticles instead of microparticles arenowadays preferentially used. Although these metal or metal oxideparticles do not give any ‘‘matrix” peak, they have a very high melt-ing point and, thus, enhanced laser fluences have to be used totransfer them into the gas phase. This often results in enhancedanalyte fragmentation [44]. Another problem that hinders theirwider application is their limited commercial availability.

Carbon nanotubes could be also successfully used and it hasbeen shown that detection limits of certain analytes can be im-proved for one or two orders of magnitude [45]. An additionaladvantage of these materials is that they can be used to enrich cer-tain apolar analytes due to their considerable hydrophobicity. Inor-ganic materials such as silicon [46], graphite [47] and TiO2 [48]have been used to coat the surface of the MALDI plate, and no addi-tional matrices were used. Fabrication of complete MALDI platesfrom porous silicon and graphite for matrix–less analyte detectionhas also been reported [49]. Such inorganic oxides are especiallyuseful because their isoelectric points vary over a wide range fromacidic to basic. Therefore, the creation of positive or negative ions isfavored depending on the oxide choice.

Some selected applications of these matrices will be describedat the appropriate places in this review. However, inorganic matri-ces are not commonly used so far and there are only very fewapplications to phospholipids available [50]. However, metal ormetal oxide nanoparticles seem particularly promising in theimaging field because they give higher resolutions than standardsolid matrices [51]. Therefore, all these materials are assumed tohave significant future potential.

3. A survey of lipids so far investigated by MALDI-TOF MS

Clearly, biopolymers and particularly proteins or peptides de-rived thereof are so far primarily investigated by MALDI-TOF MS[52]. In this review we will deal exclusively with apolar com-pounds and particularly with lipids and phospholipids that can

be found in living organisms. As we are sure that the readers of‘‘Progress in Lipid Research” will know the related chemical struc-tures, we will pay only minor attention to this point. Our previousreview in this journal [19] contained a survey of the related chem-ical aspects and may be consulted if needed. Additionally, therewas a recent paper that gave a comprehensive survey of the classi-fications of lipids [53].

3.1. Hydrocarbons and wax esters

As already indicated above, positive ion MALDI MS is muchmore common than negative ion MALDI MS. In order to convert a(neutral) molecule into a positively-charged ion, a cation, for in-stance a proton must be added. Expectedly, this normally takesplace at sites with a high electron density, i.e. charged groups suchas phosphate or atoms with an electron lone pair such as nitrogenor oxygen.

Nevertheless, even completely apolar compounds (without anyoxygen, nitrogen or sulfur heteroatoms) such as the unsaturatedhydrocarbon squalene (the structure of which is shown in Fig. 4),a hydrocarbon containing several isoprene units, as well as its olig-omerization products can be easily analyzed by MALDI-TOF MS[54]. Squalene occurs in olive oil (about 0.8–12 g/kg) and has beenassumed to be related to the health benefits of vegetable oils andis, thus, of significant nutritional interest.

Due to the apolarity of squalene, an auxiliary reagent such assilver trifluoroacetate (AgTFA) is often added to the matrix, for in-stance, 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) MScan also be used because an additional matrix is not absolutelynecessary and does not significantly improve the spectral quality.This provides the significant advantage that there is no interfer-ence with matrix peaks at all. However, direct LD analysis is impos-sible if completely saturated hydrocarbons are to be analyzed asthese compounds lack sufficient UV absorptions.

Asphaltene fractions of crude petroleum [56] can be investi-gated in a similar way and also in this case the MALDI and theLDI spectra are of comparable quality. Additionally, applicationsof MALDI-TOF MS to polyaromatic compounds [57] have also beendescribed although the insolubilities of these molecules conferredsome problems. These problems were solved by using a new sam-ple preparation method consisting of mechanically mixing analyteand matrix without the necessity to use solvents [58]. Finally,7,7,8,8-tetracyanoquinomethane (TCNQ) was shown to possesssuperior properties in comparison to other matrices [59]. A morecomprehensive survey about LD MS is available in [60] and this re-views covers also aspects of desorption/ionization on silicon (DIOS)as well as carbon-based microstructures. Finally, a quite old, butnevertheless excellent review dealing with LD MS is available in[61].


It should be noted that the lithium salt of DHB is a more suitablematrix for hydrocarbon analysis than the free acid DHB: LiDHBprovides 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 saturatedand unsaturated hydrocarbons with more than 70 carbon atomscould be detected. In a very new paper it was shown that the dis-tribution of different wax esters and other apolar compounds canalso be investigated by MALDI imaging MS [65]. Of course, thereare also many different apolar polymers the analysis of whichmight be of significant interest. These compounds will not be dis-cussed here but the interested reader is referred to the excellentreview by Batoy and coworkers [66].

3.2. Plant pigments

Since a pioneering MALDI work in 1996 [67], only very moder-ate efforts were undertaken to study chlorophylls and other plantpigments [68]. This is somewhat surprising because these pig-ments play a very central role in the plant metabolism, particularlyregarding photosynthesis. Using standard DHB matrix, such mole-cules are easily detectable. However, they are only detectable sub-sequent to removal of the central metal ion [69]. Chlorophyll a, forinstance, is detected at m/z = 871.5, although its monoisotopicmass is 892.5 (for chemical structure see Fig. 5).

The observed mass difference may be explained by the additionof three H+ and the loss of Mg2+ leaving a single positively-chargedion. A very recent study [70] provided evidence that the loss of thecentral ion can be avoided if terthiophene is used as the MALDImatrix. In contrast, however, fragmentation of the phytol-esterlinkage was more pronounced in the presence of the terthiophenematrix. It seems likely that an enhanced laser fluence was neededunder these conditions due to the weaker UV absorption of thismatrix resulting in enhanced fragmentation.

3.3. Flavonoids and carotenoids

These compounds can be easily characterized by MALDI-TOFMS although their tendency to give fragment ions seems stronglydependent on the applied matrix. According to current knowledge,

Fig. 5. Chemical structure of chlorophyll, a very important plant constituent thatenables light fixation during the photosynthesis process.

only a minor extent of fragmentation is detectable if 20,40,60-trihy-droxyacetophenone (THA) is used as matrix, whereas DHB givesmuch higher yields of fragment ions and is, thus, the matrix ofchoice to record post source decay (PSD) mass spectra [71]. Itwas also reported in the context of red wine analysis that quanti-tative 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 pos-sible [73]: as no matrix addition is required (that determines theachievable resolution due to the size of the crystals), highly re-solved (resolution about 10 lm) MS images can be obtained andit could be shown that the highly specific distribution of importantflavonoids such as kaempferol, quercetin and isorhamnetin can beimaged at the cellular level. Similar data can be also obtained if col-loidal graphite is used as matrix [74].

In a very recent work it could be shown that flavonoids such asquercetin or rutin may be used themselves as matrices for inor-ganic metal complexes [75] and provide better results in compar-ison to common crystalline matrices such as DHB.

3.4. Free fatty acids

The MALDI-TOF MS analysis of free fatty acids is quite difficult ifstandard MALDI matrices are used. The most serious problem is theoverlap of the signals of the free fatty acids with matrix signals thatare particularly abundant in the low mass range. Of course, this is aserious problem if fatty acids at low concentrations have to be ana-lyzed. According to our best knowledge standard matrices such asDHB or CHCA (a-cyano-4-hydroxycinnamic acid) are not suitablefor this purpose, but some methods as to how this problem canbe overcome have been suggested:

1. If meso-tetrakis(pentafluorophenyl)porphyrin (MTPFPP), thestructure 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 signalsbelow m/z ffi 500 in the positive-ion mode. Thus, fatty acidswith typical masses between about 200 and 350 Da can beunequivocally detected. This was demonstrated, for instance,with fatty acid mixtures obtained from different vegetable oilssubsequent to alkaline hydrolysis [77]. As an excess of sodiumacetate was added, exclusively the Na+ adducts of the Na+ saltsof the released fatty acids were detected. Therefore, there are noproblems with the overlap of different adducts, on the onehand, and differences in fatty acyl compositions (the H+ adductof arachidonic acid (20:4) results in the same m/z ratio as theNa+ adduct of oleic acid (18:1)), on the other hand, could becompletely avoided. This method was also successfully usedfor the analysis of complex fatty acid mixtures from differentbiological samples, for instance, rat plasma [78]. However,MTPFPP is so far not widely used because this matrix gives riseto unknown artifacts. Typical examples of positive ion massspectra of selected free (saturated (6a) as well as unsaturated(6b)) fatty acids recorded in the presence of MTPFPP are shownin Fig. 6. Although all saturated fatty acids are detected with theexpected m/z ratios, a mass shift of 14 amu to higher masses isobserved if unsaturated fatty acids are investigated [79]. Forinstance, oleic acid is observed at m/z = 327.2 (Na+ adduct ofthe sodium salt) as well as at m/z = 341.2. Although this effecthas not been carefully investigated yet, the above data mightindicate the oxidation of a methylene into a carbonyl group.Additionally, the sensitivity of this MTPFPP matrix is ratherpoor 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


alkaline matrix is used. 9-Aminoacridine (9-AA) seems aparticularly promising matrix in this field due to its consider-able basicity (the pKa is about 9.99) [80]. Another advantageof 9-AA is the very moderate background [81] provided bythis matrix. It could be shown that the achievable sensitivityis in the femtomolar range and linear detector responsecould be obtained over two orders of magnitude. Therefore,9-AA seems the matrix of choice for quantitative metabolomicsstudies of negatively charged compounds – not only fatty acids[82].

3. Very recently [83], it could be shown that 1,8-bis(dimethyl-amino)naphthalene (DMAN), a ‘‘superbasic” compound with apKa of 12.21 [84] is a powerful matrix because it enables thedetection of fatty acids (saturated and unsaturated ones) inlow picomole amounts. Therefore, both, 9-AA and DMAN seemmore suitable than MTPFPP because all fatty acids can be easilyand accurately detected, while the MTPFPP porphyrin matrix isobviously less suitable for unsaturated fatty acids (cf. Fig. 6b)due to the +14 amu artifact and the lower achievable sensitiv-ity. However, there are rumors that the use of DMAN maydecrease the sensitivity in the positive-ion mode significantly:residual amounts of DMAN may bind all available protons andreduce the achievable sensitivities by reducing the availableamount of this cationizing species.

4. Free fatty acids are also detectable if inorganic matrices such asgraphite [85] or porous silicon [86] (desorption/ionization onporous silicon (DIOS MS)) are used. Although the achievablesensitivity is rather poor, deprotonated fatty acids could be eas-ily detected as negative ions. A more detailed survey of thistopic is available in [87].

Fig. 6. Positive ion MALDI-TOF mass spectra of mixtures of fatty acids using MTPFPP as minvestigated, whereas in (b) a mixture of oleic (18:1), linoleic (18:2) and a-linolenic aciJournal of Food Lipids, 9 (2002) 185–200 [79].

3.5. Cholesterol and cholesteryl esters

Although cholesterol is present in virtually all mammalian cellsand body fluids [43] as well as in combination with significantamounts of cholesteryl esters (CE) and triacylglycerols (TAG) inthe lipoproteins of blood, only little interest has been paid to theMALDI MS characterization of these molecules. One potential rea-son is the commercial availability of enzymatic test kits that helpto determine both, cholesterol and cholesteryl ester concentrationsmaking MS methodology less important for the determination ofthese molecules. Using established MALDI matrices such as DHB,cholesterol is not detectable as the expected H+ adduct but onlysubsequent to water elimination at m/z = 369.3 (M+H+�H2O)[88]. Although it has been shown that the cholesterol concentra-tion in extracts of, e.g. human lipoproteins can be accurately deter-mined by MALDI-TOF MS [89], the relatively small mass ofcholesterol confers problems: in the same manner as in the caseof free fatty acids, there is a considerable overlap between the cho-lesterol peak and the matrix background. Of course, this is a partic-ularly important problem if diluted samples have to be analyzed.To our best knowledge, there were so far no attempts to establisha more suitable matrix for cholesterol analysis. In particular it hasnot yet been clarified, whether 9-AA represents a useful alternativematrix: 9-AA normally detects only charged lipids such as phos-pholipids, while an additional cationizing reagent (e.g. sodium ace-tate) is required to generate ions from molecules that do notpossess charged functional groups. These aspects will be discussedbelow in more detail and it will be outlined that the combination ofdifferent matrices represents the method of choice to obtain acomplete data set [90].

atrix. In (a) a mixture of lauric (12:0), myristic (14:0) and palmitic acid (16:0) wasd (18:3) was applied. Reprinted with permission and with slight modification from


There have been surprisingly few attempts to investigate cho-lesteryl esters by MALDI MS [89,91]: in the same way as observedfor di- and triacylglycerols (see below), CEs are exclusively detect-able as adducts with alkali metal ions (particularly sodium),whereas the H+ adducts are not detectable at all. Although notyet carefully investigated, this might be caused by the same reasonas described for triacylglycerols [92]: H+ adducts of cholesteryl es-ters are less stable in comparison to the Na+ adducts and do not‘‘survive” the flight distance to the mass analyzer of the MALDI de-vice without fragmentation. The experimental fact that the MALDImass spectra of chromatographically pure CEs always exhibit asmall cholesterol peak is a strong confirmation of this mechanism.

3.6. Sphingolipids

Sphingolipids and glycosphingolipids are currently a hot re-search topic because these compounds may be regarded to beindicative of ageing and as disease markers. As there are somerecent reviews dealing with the subject of ‘‘sphingolipidomics”[93,94], we will deal here only rather shortly with these com-pounds but will discuss this topic in more detail in the context ofTLC/MALDI (see below). Here, we will focus primarily on the anal-ysis of the most abundant sphingolipid, sphingomyelin (SM), thestructure of which is shown in Fig. 7.

SM is detectable in the same way as phospholipids and H+ as wellas Na+ adducts occur. Very recently a combination between MALDIand ESI MS was used to study changes of the sphingolipid composi-tion of parasitic nematodes [95]. It could also be proven that changesof the lipid composition of tissues (and particularly of brain) oftenaffect much more the sphingolipids than the glycerophospholipids[96]. Additionally, SM is an important constituent of virtually allbody fluids as well as tissues and, thus, particularly important inthe context of imaging studies of tissues [97]. Surprisingly, in com-parison to our previous review [19], only a few papers dealing withthe MALDI MS analysis of sphingolipids appeared. Nevertheless, itbecame obvious that a large variety of sphingolipids can be quanti-tatively analyzed by MALDI-TOF MS in the presence of an internalstandard [98]: with sphingosylphosphorylcholine as the internalstandard, the relative peak heights of SM and ceramide monohexo-side (CMH) could be used as a quantitative concentration measureand linearity could be obtained between 50 and 1500 ng SM as wellas 5 and 150 ng CMH content, respectively. Nevertheless, despitethe similarities of the headgroups, SM is less sensitively detectablein comparison to PC.

3.7. Glycerolipids

Triacylglycerols (where all hydroxyl groups of the glycerol areesterified with fatty acids) are very abundant in animal organisms,whereas compounds such as mono- or digalactosylglycerols areparticularly abundant in plants [99]. We will focus here primarilyon animals and only to a lesser extent on plant lipids. Therefore,characteristics of TAGs (and to a minor extent diacylglycerols) willbe primarily discussed.

Fig. 7. Chemical structure of the backbone of sphingomyelin. ‘‘R” represents avariable alkyl chain.

3.7.1. Di- and triacylglycerolsTriacylglycerols (TAG) are important for the storage of energy

(fat tissues in living organisms), while diacylglycerols (DAG) thatare normally generated from glycerophospholipids by cleavage ofthe polar headgroup under the influence of the enzyme phospholi-pase C are important lipid-derived second messengers [100]. Thepositive ion MALDI-TOF mass spectra of both, DAGs [101] as wellas TAGs [102] can be easily recorded with standard DHB. TheMALDI mass spectra give always exclusively the Na+ adducts,whereas H+ adducts are never detected, even if the solutions areacidified [92]. Additionally, there are always intense fragment ionscorresponding to the loss of one sodiated fatty acyl residue. UsingMALDI MS in combination with high energy collisionally-induceddissociation (CID), the losses of these fatty acyl residues can beused for structural elucidation of TAGs [103], i.e. the determinationwhich fatty acyl residue is located in which position. It is importantto note that exclusively the Na+ adducts were useful for that pur-pose while neither the K+ nor the Li+ adducts (that can be easilyobtained if the spectra are recorded in the presence of the corre-sponding alkali salts) gave indicative fragment ion spectra [103].

The applied matrix has only a relatively weak impact on thespectral quality, whereas strongly different sensitivities areachieved in dependence on the used solvents. For instance, CHCAand DHB dissolved in a mixture of acetonitrile and water gave onlysensitivities in the pmol range, whereas DHB in acetone provideddetection limits in the fmol range. Additionally, DHB provides aweaker background than cinnamic acid derivates [104] that tendto significant matrix cluster generation. This is even more impor-tant than the achievable sensitivities because TAGs (e.g. from veg-etable oils) are normally available in huge amounts. In order tohave highly reproducible spectra, sample preparation is veryimportant and particularly the ion content must be carefully con-trolled. For instance, it was recently shown that 9-AA detectsTAG only in the presence but not in the absence of additionalcationizing 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 convincingexplanation has been recently given [92] and is based on the obser-vation that the generation of fragment ions can be significantly re-duced under alkaline conditions. Thus, it was suggested that theobserved fragments actually arise from unseen protonated TAGsas their fragmentation occurs so rapidly and completely that pro-tonated TAGs are normally not observed. If the pH is increased,the H+ concentration is simultaneously decreased leading to re-duced H+ adduct generation and, accordingly, to a lower yield offragmentation products [92]. Very recently it has been shown thatthe yield of TAG fragments can be significantly reduced if the MAL-DI target is covered with a thin nitrocellulose film prior to deposi-tion of the TAG of interest [106]. The nitrocellulose filmsimultaneously improves the shot-to-shot and sample-to-samplereproducibility through the exhibition of a more homogeneousmatrix/analyte co-crystallization.

It has been repeatedly shown that MALDI MS is very suitable forthe screening of the compositions of crude TAG mixtures, for in-stance 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 beunequivocally assigned, bromination and hydrogenation was alsoapplied and combined with PSD fragment ion spectra [109].

Another important application of MALDI-TOF MS is the investi-gation of the changes occurring in oil samples during frying [111].This is a very important practical application because products


generated upon deep frying may include potentially hazardouscompounds [112]. Chromatographic enrichment of the polar com-pounds prevented mass spectrometric ion suppression, thus allow-ing the detection of minor species originating from thermaloxidation. Particularly, diacylglycerols (DAG), oxidized TAG as wellas TAG dimers, and TAG fragments arising from the homolyticbeta-scission of linoleyl, peroxy, and alkoxy radicals were foundto be indicative of the frying process. Recently, lipid compositionaldata determined by MALDI-TOF MS and HPLC/APCI-MS were com-pared [113]. It turned out that there were no major differencesmaking MALDI a suitable tool to investigate the lipid compositionsof mixtures.

3.7.2. GlycoglycerolipidsPlant and algal photosynthetic membranes contain relatively

small amounts of phospholipids, but high amounts of three glyco-glycerolipids [99], monogalactosyl-diacylglycerol (MGDG), digalac-tosyl-diacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol(SQDG). Due to differences in biosynthetic pathways, these lipidscontain high amounts of linoleic and, especially, alpha-linolenicacids which have to be taken in the diet of most animals.

All glycoglycerolipids of plants can be easily characterized byMALDI-TOF MS and some selected mass spectra are shown inFig. 8. Please note that the characteristic mass difference of22 amu is not caused by H+/Na+ exchange, but both peaks corre-spond to the Na+ adducts of lipids with different fatty acyl residues,for instance DGDG 34:3 and DGDG 36:6. Therefore, it is unique ofthese compounds – in the same manner as TAG and DAG that theydo never generate H+ adducts but exclusively Na+ adducts.

Please note that the discussed glycoglycerolipids can be easilyseparated by means of TLC [69]. Therefore, the relative contribu-tions of the individual lipid classes can be most easily determinedby TLC while the individual fatty acyl residues can be easily iden-tified by MALDI-TOF MS. Finally, it has been shown by PSD MALDI[114] that the fatty acyl residue in sn-1 position is preferentiallylost. This preferential loss may also be used for the determinationof the positions of the individual fatty acyl residues.

It has also been shown that MALDI MS combined with TLC is asuitable, generally applicable method of lipid analysis [22].

3.8. Glycerophospholipids

Glycerophospholipids (GPL) are the most important subclass ofglycerolipids and merit, thus, a separate chapter. GPL are not onlyof in vivo relevance as important constituents of the cellular mem-branes, but some GPL (such as phosphatidic acid, phosphoinosi-tides and lysophospholipids) are also involved in signaltransduction processes, i.e. they act as second messenger mole-cules [115].

Therefore, GPL are attracting increasing interest as potentialdisease markers in important, socioeconomically relevant diseasessuch as atherosclerosis [116] or rheumatic diseases [117]. Manydifferent chromatographic, spectroscopic as well as MS methodsto assess the GPL composition of an unknown sample (e.g. an or-ganic extract of a body fluid) are known and have been reviewed[118,119]. The schematic structures of some selected GPL thatcan normally be found in organic extracts from cells and body flu-ids and that will be primarily discussed below in this review areshown in Fig. 9.

Although ESI MS was so far primarily used in ‘‘lipidomics” stud-ies [120], the number of lipid studies making use of MALDI MS isincreasing [121]. One potential reason for the nowadays extremelygrowing interest in lipid analysis by MALDI MS is coming from‘‘MALDI MS imaging” that is currently experiencing significantinterest [122] and will be explained below in more detail. As all rel-evant tissues contain large numbers of cells and all cells possess a

membrane composed of considerable quantities of GPL and furtherapolar components such as cholesterol, lipids are easily detectablein all MALDI imaging experiments of tissues [123].

Despite the considerable technological progress that has beenachieved regarding the available MS hardware [124], aspects ofthe ‘‘optimum” MALDI matrix (if it does anyway exist) have notyet been clarified. Therefore, there is currently an increasing num-ber of papers dealing with ‘‘matrix engineering” [21,29].

3.8.1. Zwitterionic glycerophospholipids (PC and PE)Among all GPL so far investigated, phosphatidylcholine (PC) has

been by far, the most comprehensively studied. This has severalreasons:

1. PC is the most abundant constituent of cellular membranes – atleast as far animals or humans are concerned.

2. PCs are commercially available (for instance, from AVANTI PolarLipids, Alabaster, USA) with varying fatty acyl residues and,thus, can be easily used as model compounds. Additionally,alkyl- and alkenyl ether derivates are also available.

3. PC possesses a quaternary ammonia group that contains a per-manent positive charge. Therefore, PC is most sensitivelydetectable in lipid mixtures – at least if the positive-ion modeis considered.

Regarding (3), i.e. the achievable sensitivity, it is important tonote that this is not only dependent on the MALDI device usedand, thus, the available mass analyzer and detector but also signif-icantly on the applied matrix. Therefore, it is senseless to providedetection limits without specifying the applied matrix. Since DHB(2,5-dihydroxybenzoic acid) is one of the most established andbest characterized matrix compounds [125], the majority of theavailable data were obtained with this matrix [126] and some se-lected data are given in Table 1.

Table 1 clearly shows that different lipid classes exhibit verydifferent detection limits and points out that the charge state ofa lipid determines its detectability. Please note that sulfatides con-taining a negatively charged sulfate group are much less sensi-tively detectable than other lipids in the positive-ion mode.Therefore, the comparison of the positive and the negative ionmode is normally a straightforward approach and this is best doneby using different matrix compounds with different acidities, forinstance, DHB for positive ion and 9-AA for negative ion detection.This will be explained below in more detail.

The significant importance of the matrix on the quality of MAL-DI mass spectra of phospholipids was already recognized in a pio-neering study by Harvey [127]. Although a large variety of differentmatrices were tested, sinapinic acid, a-cyano-4-hydroxycinnamicacid and DHB gave the best results regarding the achievable sensi-tivity, resolution and the extent of (unwanted) fragmentation. Itwas also noted that individual PL classes are detected with extre-mely different sensitivities [127]. Particularly PE is much less sen-sitively detectable than PC in the positive ion detection mode(acidic PL such as PS are much more sensitively detectable as neg-ative ions and will not be considered here): this is due to the per-manent positive charge of the quaternary ammonia group of the PCmolecule, whereas the amino group of the PE can be easily depro-tonated leaving the negative charge of the phosphate group [128].This is the reason why the positive ion spectra of cell or tissue ex-tracts are dominated by PC signals [121]. Therefore, separation intothe individual lipid classes is normally needed in order to analyzethe detailed lipid composition of mixtures. In contrast, the pre-ferred detectability of certain lipid classes (particularly PC, LPCand SM) may also be regarded as an advantage because the massspectra are less complex [32]. The preferred detectability of PC in

Fig. 8. Positive ion MALDI-TOF mass spectra of organic solutions of commercially available MGDG (bottom), DGDG (middle) and SQDG (top). Please note that all lipidsrepresent mixtures isolated from spinach. All spectra were recorded using a 1:1 (v/v) mixture between the lipid of interest (1 mg/ml) and the matrix (0.5 M DHB in methanol).About 1 lg lipid was used per sample. All peaks are labeled according to their m/z ratios. Reprinted with permission and modification from Chemistry and Physics of Lipids150 (2007) 143–155 [69].

Fig. 9. Chemical structures of headgroups of selected, abundant glycerophospholipids. Only the characteristic headgroups (abbreviated by ‘‘X”) are shown while ‘‘R” denotes astrongly varying acyl residue. Please note that the majority of physiologically relevant lipids possess a saturated fatty acyl moiety at the sn-1 position and an unsaturated fattyacyl residue at the sn-2 position. Please also note that in addition to ester-linked compounds, there are also alkyl-acyl as well as alkenyl-acyl (termed ‘‘plasmalogens”)compounds.



comparison to PE is illustrated in Fig. 10 using an extract from henegg yolk.

It is obvious from Fig. 10, that even if the PE is easily detectableper se in the absence of PC, not even small PE peaks are detected ifa total egg yolk extract is investigated without previous separationinto the individual lipid classes. Therefore, mixture analysis shouldbe always regarded with great caution.

This is a particular problem in the case of PE because this PL isnot (or only with very low sensitivity) detected as negative ion ifclassical acidic matrices such as DHB are used [129].

Using matrices such as para-nitroaniline (PNA) [130] or 9-ami-noacridine (9-AA) [105], this problem can be easily overcome.However, it has been shown very recently that PC is also detectablein the presence of 9-AA as negative ion [131] subsequent to theloss of a methyl group and this is exemplarily shown in Fig. 11.

Fig. 11 provides clear evidence that DHB and 9-AA result incharacteristic differences. There are obviously only slight differ-ences between DHB and 9-AA in the positive-ion mode, althoughthe ratio between the H+ and Na+ adduct (e.g. m/z = 760.6 and782.6 in the case of POPC, 11a,b) depends on the matrix and 9-AA favors the generation of H+ adducts [105]. However, the nega-tive ion mode spectra differ more significantly. For instance, a sin-gle signal is obtained if POPE is investigated in the presence of 9-

Table 1Survey of relative detectabilities and the related detection limits of differentimportant lipid classes. All data were recorded on a standard MALDI-TOF instrumentin the positive-ion mode using the reflector mode and DHB as matrix. Data weretaken from [126].

Lipid class Relative detectability (%) Detection limit (ng)

PC 100 0.020PS 70 0.029Sphingomyelin 70 0.029PG 40 0.050Galactocerebrosides 40 0.050PI 20 0.100PE 2 1.000Sulfatides 0.001 2000

Fig. 10. Positive ion MALDI-TOF mass spectra of an organic extract (obtained accordingspectrum of the total extract, whereas trace (b) corresponds to the PC fraction and traceSeparation of lipids was performed by TLC prior to MS analysis. Although some further TThe dotted lines indicate the peaks of the most abundant PE species, PE 16:0/20:4.

AA (11f), while many different signals (primarily stemming fromthe DHB matrix [104]) are observed in the presence of DHB(11e). Differences are still more obvious if the negative ion spectraof POPC are compared: the signal at m/z = 744.6 in the negative ionmode is caused by a loss of one methyl group from the PC (11h),whereas only a cluster ion between DHB and POPC can be observedin (11g) at m/z = 912.6 [132]. Although not yet investigated in de-tail, it is evident that PC may be easily misinterpreted as PE (andvice versa) if negative ion spectra are recorded.

Although the loss of a methyl group from PC is rather commonin the case of ESI MS, this mechanism was so far only described asingle time regarding MALDI MS [133]: Marto and coworkers haveshown that PC is (in addition to its detection as positive ion) alsodetectable as negative ion if trans-4-hydroxy-3-methoxycinnamicacid is used as matrix. In addition to the loss of a methyl group[M-15]�, loss of a quaternary amine [M-60]� and loss of the cholineheadgroup [M-86]� could be simultaneously detected.

Although the discussion of the detectability of different lipidseither as positive or negative ions seems to be of academic rele-vance only, this is absolutely not true: being able to detect a PLas negative ion, makes the determination of its fatty acyl composi-tion much simpler. For instance, a certain PL containing one stea-royl and one linoleoyl acyl residue, on the one hand, and twooleoyl residues, on the other hand, result in exactly the samemasses. Using MS/MS, however, this problem can be easily solvedby monitoring the loss of the individual fatty acyl residues. As fattyacids are more sensitively detectable as negative ions, it is muchbetter to use the negative ion detection mode but this is only appli-cable if the parent ion gives a clearly detectable negative ion signal.For a more detailed discussion of MALDI fragment ion spectra ofPLs see [114].

Although DHB is so far most established, this compound issurely not the most sensitive matrix: using clathrate nanostruc-tures (i.e. an inorganic matrix), it could be recently shown thatlysophosphatidylcholine (LPC) is already detectable if it is presentin a concentration of about 7 � 10�22 mol. As 1 mol is equivalent to6 � 1023 molecules this indicates that already about 10 moleculesof the analyte are sufficient to obtain a detectable signal [134].

to the method by Bligh and Dyer) of hen egg yolk. Trace (a) represents the mass(c) to the PE fraction, respectively. All spectra were recorded with DHB as matrix.

LC fractions could be obtained, only the most relevant lipid classes are shown here.

550 600 650 700 750 800 850 900m/z [Th]m/z [Th]

550 600 650 700 750 800 850 900

(a) (b)

(d)(c)

(f)(e)

(h)(g)

O P O

O

O

CH2 CH2 N CH3

CH3

CH3

O P O

O

O

CH2 CH2 N CH3

CH3

CH3

O P O

O

O

CH2 CH2 N H

H

H

O P O

O

O

CH2 CH2 N H

H

H

N

NH2

HO

OHCOOH

681 (*)

857 (*)

681 (*)

675 (*)

912.6

716.5752.5 782.5

892.5

762.5

762.5740.5 740.5

716.5

744.6

754.6

821.5

718.5577.5 603.5

630.5

650.5672.5

760.6 760.6

782.6

782.6

706585.0

673.4 699.4

Fig. 11. Positive and negative ion MALDI-TOF mass spectra of 1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC; a,b,g,h) and 1-palmitoyl-2-oleoyl-sn-phosphatidyleth-anolamine (POPE; c,d,e,f). In all cases a 0.5 M solution of DHB in methanol (left; a,c,e,g) or 9-AA in isopropanol/acetonitrile (right; b,d,f,h) were used as matrices and thesample solutions (0.2 mg/ml) diluted 1:1 (v/v) with the matrix. All peaks are marked according to their m/z ratio and the DHB matrix peaks are marked with asterisks. Thepolarities of the performed measurements and the headgroups of the investigated lipid species are indicated in the figure. Reprinted with modification and permission fromAnal. Bioanal. Chem. 395 (2009) 2479–2487.


Although this approach is surely not broadly applicable in practice,this low detection limit sheds light on the high sensitivities ofmodern MS devices and particularly their detectors.

The high sensitivity for detecting PC is obviously an advantage ifderivatives of PC (i.e. with the same headgroup) are of interest. Thisparticularly concerns LPC that lacks in comparison to PC one fattyacyl residue – normally in sn-2 position [135] – and the PC/LPC ra-tio has been proven to be useful as disease marker for instance ofrheumatic diseases [117]. It has also been shown that LPC is notonly generated by the (most widely established) enzyme phospho-lipase A2 (PLA2) but as well under the influence of reactive oxygenspecies (ROS) such as HOCl [136]: in the first step of the reaction,HOCl is added to one (or several) double bonds of the PC to gener-ate the corresponding chlorohydrins. The characteristic mass shiftof 52 amu can be easily monitored by MS. The introduction of thesestrongly electronegative atoms (chlorine and oxygen) leads to thereduction of the strength of the ester bond making hydrolysis ofthis bond easier and finally to the generation of LPC [137]. Thus,LPC is obviously a marker of inflammatory conditions accompaniedby oxidative stress. Of course, in addition to HOCl there are manymore ROS that are generated under inflammatory conditions lead-ing to different lipid oxidation products [138].

Although oxidation products such as endo- or hydro-peroxidesare also basically detectable by MALDI-TOF MS, they are only de-tected with much lower intensities [139,140]. Even if the detailedreasons are not yet known, it seems reasonable to assume that la-ser irradiation leads (at least) to partial degradation of these ini-tially formed products under generation of carbonyl compoundsupon cleavage of the original double bond. Very recently 6-aza-2-thiothymine was suggested as the matrix of choice to detect oxi-dized phospholipids [141]. A more detailed review dealing with

the mass spectrometric detection of oxidatively modified lipids isavailable in [142].

Unfortunately, MALDI-TOF MS investigation of the products ofthe reaction between HOCl and PE is much more difficult: PE con-tains, in comparison to PC, an additional reactive functional group,the amino group that exhibits a higher reactivity in comparison tothe olefinic residues. Therefore, the prime reaction products aremono- as well as dichloroamines. It was shown that these productscan be detected by electrospray MS but not by MALDI-TOF MS be-cause decomposition occurs rapidly [143]. However, this onlyholds if standard matrices such as DHB or CHCA are used. In con-trast, the expected products are easily detectable if a chlorinatedcinnamic acid (4-chloro-a-cyanocinnamic acid) derivate is used[144] and the corresponding positive ion MALDI mass spectra areshown in Fig. 12.

The underlying mechanisms for these surprising differencewere discussed in detail and related to the different proton affini-ties of the individual matrices in [145]. This example proves clearlythat matrix design in order to develop more suitable matrices isvery important and not only of academic interest.

Finally, in addition to diacyl PLs there are also lipids with alke-nyl ether bonds and these compounds are commonly termed‘‘plasmalogens” [146]. Although these compounds can be analyzedin exactly the same manner as common PCs, there is one importantdifference: plasmalogens are extremely sensitive towards eventraces of acids that leads to the cleavage of the alkenyl ether link-age under generation of the corresponding LPC. Although the acid-ity of MALDI matrices such as DHB is tolerated, the use of TFA(trifluoroacetic acid) as an cationizing agent is strongly discour-aged because TFA in a concentration of about 0.1% is already suffi-cient to induce partial hydrolysis of plasmalogens [147]. This is a

(a)

(b)

(c)

680 700 720 740 760 780 800 820

m/z[Th]

692.5

692.5

714.5

714.5

784.5

806.5

714.5

736.5

736.5

782.5

804.5

736.5

Fig. 12. Positive ion mode MALDI-TOF mass spectra of 1,2-dipalmitoyl-sn-phosphatidylethanolamine (DPPE) after treatment with a 10-fold excess of HOCl. Spectra wererecorded with different matrices: DHB (a), CHCA (b), and ClCCA (c) were used. Please note the exclusive detectability of the chloramine of DPPE (at m/z = 782.5 and 804.5corresponding to the H+ and the Na+ adduct, respectively) in the case of ClCCA. In all other cases only DPPE is detected as H+ (m/z = 692.5), Na+ (m/z = 714.5) and �H++2Na+

adduct (m/z = 736.5). Reprinted with modification and permission from J. Am. Soc. Mass Spectrom. 2009, 20, 867–874 [145].


particular problem, if spermatozoa and brain tissue samples are ofinterest because these are characterized by high plasmalogen con-tents [146].

3.8.2. Acidic glycerophospholipidsAs expected, phospholipids such as PI, PS and PA are detectable

with rather poor sensitivities as positive ions due to their negativecharge [126]. Additionally, these lipids occur normally in rathersmall amounts and, thus, they are rather difficult to detect. As theyare not commercially available to such an extent as PC and PE andare often much more expensive, they have been much less ofteninvestigated although they often have higher biological relevancethan the bulk phospholipids, PC and PE.

This particularly concerns phosphorylated phosphoinositides(PPI) that possess three or more negative charges due to their addi-tional phosphate residues [148]: all phosphoinositides produceexcellent signals in the negative ion mode when analyzed as iso-lated compounds in amounts of about 1 nmol. However, the detec-tion thresholds for phosphoinositides are higher than that of moreabundant PLs and increase further with the phosphorylation state.While PC, for instance, is detectable in amounts of less than onepmol, the amount of PIP3 on the sample plate that is needed to pro-duce a sufficient signal is higher than 700 pmol [149]. Similar datawere also reported regarding the detection of cardiolipin [150] thatis also more highly charged than common PLs. Beside the charge, itmight also be possible that the increased polarities of these com-pounds lead to reduced interactions with the matrix that is crucialfor the ionization process. However, comprehensive investigationswhy phosphoinositides and cardiolipins are only scarcely detect-able by MALDI MS are not available so far. This is a pity becausethese compounds are particularly important, functional molecules.

Recently it has been shown [151] that PI species can be easilymonitored and even quantified in rather complex mixtures by

MALDI-TOF MS subsequent to the removal of interfering PC byits binding to a small column filled with a silica gel cation exchan-ger (SCX). A more comprehensive review of this important topic isavailable in [152].

Although PS is a lipid that is of high diagnostic relevance partic-ularly in the context of assessment of apoptotic cells [153], surpris-ingly few attempts have been made to characterize PS by MALDI-TOF MS. To our best knowledge there are just a very few papersdealing with this topic: MALDI-TOF MS was recently used to inves-tigate the lipid composition of tear samples from normal and dryeyes of rabbits [154], whereby a solid ionic crystal MALDI matrixof para-nitroaniline and butyric acid was used that gave superiorresults in comparison to common crystalline matrices. In additionto an enhanced SM content in the dry eye tears, PS species werereadily only detectable in dry eye tears but not in the normal tears.Using artificial PL membranes, the influence of HOCl on PS wasinvestigated and many different products were detectable. As al-ready discussed above, chlorinated compounds are only detectableif 4-chloro-a-cyano-cinnamic acid is used as a matrix [155] but notin the presence of standard matrices.

The detection of phosphatidic acid (PA) and derived lyso-phos-phatidic acid (LPA) is a quite challenging task for the same reasonsas the phosphoinositides: PA and LPA possess a high negativecharge density and are, thus, much less sensitively detectable thancommon zwitterionic phospholipids. Additionally, PA and LPA areonly present in very small amounts in biological samples. In orderto overcome these problems, an inorganic zinc complex ([1,3-bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato] dizinc(II)) wasused as matrix by Tanaka and coworkers [156] in order to trapthe LPA and to significantly enhance its detectability. 0.1 pmol ofLPA 18:1 was detectable on the MALDI target under these condi-tions. This method was very recently improved and extended tosphingosine-1-phosphate (S1P) in order to make this approach


applicable to high throughput screening of samples with signifi-cant clinical interest [157].

4. Analysis of lipid mixtures

The different detection limits of the individual PL classes as de-scribed above and exemplarily reviewed in [126] are extremelyimportant for the analysis of biological samples: if crude organic ex-tracts from cell cultures, body fluids or tissues are to be investigatedby MALDI-TOF MS without previous chromatographic purification,it may happen – depending on the composition of the sample – thatonly some selected lipid classes, particularly PL with quaternaryammonia groups are detected while the remaining ones are sup-pressed [158]. It is also a matter of fact that the different lipid classesinfluence the intensities of others and it is often observed thatMALDI mass spectra change if they are recorded at different analyteconcentrations. This is illustrated with an extract from egg yolk as atypical example (Fig. 13): it is evident that spectra change signifi-cantly with increasing dilution and particularly the apolar compo-nents, i.e. the triacylglycerols (TAG) are only detectable if dilutedsolutions are investigated. Such suppression effects are well knownin MALDI-TOF MS and were thoroughly studied regarding the sup-pression of matrix signals [159] as well as analyte signals [160].Please also note that the ratio between the H+ (m/z = 760.6) andthe Na+ (m/z = 782.6) adduct of PC 16:0/18:1 is not constant butthe Na+ adduct is obviously favored with increasing dilution. A moredetailed discussion of these effects is beyond the scope of this paper,but can be summarized as follows: when more analyte is presentthan primary ions, suppression occurs as a consequence of second-ary reactions and this effect is also significantly dependent on theapplied laser fluence. It is well known to each MALDI user that thereis a direct relation between signal intensity and analyte concentra-tion only in a small concentration range, while the achievable signalintensity decreases at higher concentrations. If the applied amountof sample exceeds a certain limit, no signal is detectable anymore.This behavior has been recently explicitly described [161]. There-fore, it is highly recommended to use similarly concentrated solu-tions of the analytes and to perform all measurements in thepresence of an internal standard.

Fig. 13. Positive ion MALDI-TOF mass spectra of an organic extract from hen egg yolk deprior to deposition onto the MALDI target. The egg yolk extract was diluted with CHCl3

According to our experience there is no absolute need to use anisotopically labeled reference compound, but a lipid with oddnumbered fatty acyl lengths may also be used. So far, there is noagreement that a special standard must be used for each lipid class,or if a single compound (each for positive and negative ion detec-tion) is sufficient. Although relative peak intensity ratios may alsobe used, this method does not allow one to judge if both or onlyone concentration are changing and, thus, the use of internal stan-dards is recommended.

It is also highly recommended that the positive ion spectra arerecorded in the presence of an acidic (e.g. DHB) and the negativeion spectra in the presence of an alkaline (e.g. 9-AA) matrix. IfDHB, the most common matrix in lipid analysis is used, PC andother PLs with quaternary ammonia groups such as LPC or SM,are detectable as positive ions, whereas negatively charged PLssuch as PI and PS are only detectable as negative ions or justwith very low sensitivity in the positive ion detection mode.Using careful sample preparation, quantitative data regardingthe compositions of different cells [105], lipoproteins [89] as wellas meat [162] could be obtained. These are only some selectedexamples but MALDI MS can surely be used to obtain quantita-tive data. A more comprehensive survey of quantitative aspectsof MALDI MS regarding small molecule analysis is available in[163].

There is another important aspect beside mass spectrometricmeasurements: the influence of extraction on the lipid yield. Mix-tures consisting of chloroform and methanol are the most estab-lished and used in the majority of cases [118]. Although thistopic would be worth its own review, the following critical aspectsshould be considered:

1. Enrichment of lipids by extraction with chloroform (accordingto the methods developed by Bligh and Dyer [164] or Folchet al. [165]) works quite well for bulk lipids such as PC or PEand these lipids are nearly quantitatively extracted. However,more polar compounds such as LPA or PPI are incompletelyextracted because these compounds have a higher affinity toremain in the water/methanol phase. If these molecules are ofprime interest more polar solvent mixtures and/or acidifyingthe solution is required.

pending on the amount of sample. All samples were mixed 1:1 with matrix solutionin several steps as indicated in the figure.


2. Surprisingly, apolar lipids are also incompletely extracted ifstandard methods such as the Bligh and Dyer approach [164]are used and more apolar conditions should be used for thatpurpose. The reason for this incomplete extraction is mostprobably the tendency of apolar lipids to form other structuresthan amphiphilic PL that provide well-defined supramolecularstructures.

3. If an aqueous sample containing proteins is treated withorganic solvents, precipitation of proteins and other highlypolar compounds will occur. This is obvious by the generationof a white ring at the interface between the aqueous and theorganic phase. Some lipids may stick to the precipitated pro-teins and are, thus, lost if only the organic layer is used for fur-ther analysis.

In order to make a long story short: there is obviously not a sin-gle method that allows the unequivocal extraction of all PL classesand great care is required if absolute, quantitative data are re-quired. There was one 31P NMR based study that has comparedthe use of detergents and organic solvents for the (lipid) extractionof human erythrocytes [166]. It was one result of this study thatextraction with the detergent sodium cholate in comparison to or-ganic solvents resulted in a much higher yield of PLs and would,thus, be the method of choice in order to extract lipids quantita-tively. Unfortunately, this method is only applicable to 31P NMRwhere the presence of the detergents does not play a role becausethey are not detected at all. In contrast, interference has beenshown recently following the treatment of boar spermatozoa withdifferent detergents and subsequently assessing the lipid composi-tions of the extracts by MALDI-TOF MS: in the presence of evensmall amounts of detergents, the sensitivities for lipid detectionwere strongly reduced [167]. Thus, the potential presence of deter-gents is a considerable problem: detergent removal kits are avail-able for proteins, but there is, so far, no commercial detergentremoval kit for the organic (lipid containing) phase. This is caused(a) by the lower interest that lipids were so far experiencing and(b) the fact that most detergents exhibit a significant distributionof their molecular weights. This makes their removal much moredifficult in comparison to compounds with a defined molecularweight. In order to illustrate this fact in Fig. 14 the positive ionMALDI-TOF mass spectrum of a commercially available TWEEN

Fig. 14. Positive ion MALDI mass spectrum of commercially available TWEEN 20, a commDHB as matrix. Please note the considerable distribution of the masses due to variabledifferent groups of peaks that are stemming from different structures. These are shown

20 preparation is shown: although we do not want to discuss thisspectrum in detail (further details are available in [167,168]), it isevident that the spectrum of this detergent is very complex andcovers a significant mass range. This makes detergent removalrather difficult and, therefore, the application of detergents to ex-tract lipids from biological sources seems not to be the methodof choice. Nevertheless, there are some papers dealing with this ap-proach and the use of dodecyl maltoside for membrane solubiliza-tion seems preferential because this detergent exhibits a lowerextent of heterogeneity [169]. The reader should also note thatparameters such as the salt concentration influence the achievablespectral quality although this is a much smaller problem in thecase of lipids because the majority of the salt is already removedby the extraction process [170]. Nevertheless, in order to accountfor different salt concentrations, all adducts (normally the H+ andNa+ adduct) of a selected lipid must be used for subsequent dataanalysis. This means that the combined intensities of both adductsmust be used for proper quantitative analysis.

Regarding mixture analysis, one should also wonder if completeinformation is really always needed. Would less sometimes bemore? This particularly holds because the difficulties of properdata analysis often strongly increase as more data are collected.According to our opinion it is often sufficient to have some definedselected metabolites determined. For instance, we have establishedthe PC/LPC ratio as a measure of the quality of a human ejaculate[171] that correlates very well with the amount of annexin-V-po-sitive spermatozoa. Very recently, we have also shown that thisis not only possible for human spermatozoa but may also be ap-plied to the analysis of animal spermatozoa, for instance from catsand cattle [172]. The structures of the related molecules are shownin Fig. 15. Although it is not yet clear whether (a) increased activityof PLA2 or (b) an enhanced generation of ROS or (c) a reducedreacylation of the LPL primarily contribute to the changed PC/LPCratio, this ratio seems a suitable measure of the quality of sperma-tozoa. A very simple method to quantitatively extract LPC fromaqueous samples that uses only methanol and necessitates only asingle centrifugation step was suggested recently [173].

Somewhat related to spermatozoa research, it was very recentlyshown that different animal oocytes provide very different MALDImass spectra and can be, thus, easily differentiated [174] and givemolecular fingerprints of the lipid composition.

on detergent for the solubilization of cellular lipids. The spectrum was recorded withnumbers of bismethylene oxy groups. Please also note that there are at least twoin the upper part of the figure.


5. Separation of the individual lipid classes prior to analysis

When separated, all phospholipid classes (if present in suffi-cient amounts) are easily detectable by MALDI MS because sup-pression effects between the individual lipid species do not playany role. Nevertheless, detection limits for the individual lipid clas-ses [126] are very different due to the reasons discussed above.

Separation into the different lipid classes may be of course per-formed by offline HPLC and the fractions obtained subsequentlyanalyzed by MALDI-TOF MS. We will not pay major attention tothis application because HPLC is normally not coupled to a MALDIbut to an electrospray ion source and this important method of li-pid analysis has been already comprehensively reviewed recently[175]. This also holds for the use of, for instance, APCI instead ofESI [176].

In contrast, we will focus here on thin-layer chromatography(TLC), that is even nowadays still a widely used separation methodin lipid research [177] due to its simple and fast (several samplescan be separated at the same time) performance as well as the verymoderate price of the required equipment. TLC is also very ecolog-ically-friendly because much lower amounts of solvents areneeded in comparison to HPLC. Finally, certification of TLC separa-tions is simpler in comparison to HPLC: using TLC, a completelynew stationary phase is always used, whereby potential contami-nations from a previous run can be surely avoided, while in thecontext of HPLC one may always suspect that there is some resid-ual amount of the analyte even subsequent to intense washing ofthe column [178].

Clearly, TLC separation may be also offline coupled to MALDI:the lipid mixture of interest is separated by TLC and afterwardsstained by a non-destructive dye that does not lead to alterationsof the masses of the lipids of interest. This requirement is fulfilled,for instance, by the fluorescent dye primuline that binds non-cova-lently to the fatty acyl residues of lipids [179] and can be easily re-

Fig. 15. Survey of the conversion of PC into LPC under the influence of the enzyme PLA2

while a high plasmalogen content is characteristic of many animal spermatozoa. Accordiexhibit different stabilities.

moved by changing the polarity of the solvents. Afterwards thesilica gel with the attached lipid is scraped off from the TLC plate,the lipid is eluted by organic solvents and subsequently analyzedby MALDI MS. Some selected examples of this technique are theanalysis of characteristic lipid oxidation products [180], cells suchas bull [147] or human spermatozoa [181], body fluids such as lungsurfactant [182], tissues such as brain [183] or organic extractsfrom algae [69].

Although this approach is capable of providing convincing re-sults, it is obviously boring and time-consuming, particularly ifmany samples each containing a lot of different lipid species haveto be analyzed. Additionally, this method bears the risk of losingcertain lipid species. For instance, it has been shown that an ‘‘effectof chromatography” exists [184]. This means that lipids with dif-ferent fatty acyl residues are differently tightly bound to the silicagel and are, thus, eluted to a different extent. Therefore, the fattyacyl compositions monitored prior and subsequent to chromatog-raphy may be different [184].

Despite these problems, a method enabling a more direct cou-pling between TLC and ESI MS was recently successfully estab-lished [185]. A plunger based extraction interface (nowcommercially available as the ‘‘ChromXtract” from the CAMAGcompany) combined with an HPLC pump was shown to providegood results for quantitative TLC/ESI MS from HPTLC silica gelplates regarding repeatability of the MS spectra and the achievablelimit of detection (LOD) and the limit of quantification (LOQ). Thiswas shown using harmane, a heterocyclic aromatic amine, as theselected model compound [186]. Modifications of the deviceenabled complete, highly reproducible extraction of analytesfrom glass backed as well as aluminum backed TLC and HPTLCplates, layers with thicknesses up to 100 lm and different station-ary phases [187]. This application is surely a current ‘‘hot” topic[188,189] and of interest for scientists working in differentfields.

or ROS. Please note that human spermatozoa are particularly rich in PC 16:0/22:6,ngly, different products may be expected because the acyl- and the alkenyl-linkage


Since a ‘‘solid” sample is used in TLC, i.e. the lipid is dispersed ina solid silica gel ‘‘matrix”, the idea to combine MALDI directly withTLC seems a straightforward approach and was already attemptedat the end of the 1990s [190]. MALDI characterization of the devel-oped TLC plate would offer the advantage of much higher resolu-tion because the achievable resolution is determined primarilyby the laser spot size that is normally of the order of only about20 lm. This means that 50 individual MALDI mass spectra can berecorded from a TLC spot of a diameter of 1 mm and provides thepossibility to resolve different components that could never be re-solved if the complete spot is scraped from the TLC plate. However,there are three important problems that must be solved [22]:

1. The matrix must be supplied in a very homogeneous fashionand in the form of very small droplets since otherwise theachievable resolution would be compromised by the spreadingof the matrix solution on the TLC plate as well as the formationof large matrix–analyte crystals.

2. The majority of commercially available MALDI-TOF devices arecurrently equipped with UV lasers (k = 337 nm). UV irradiationdoes not penetrate very deeply into the silica layer and, thus,primarily compounds near to the surface of the TLC plate aredetected. Due to these problems, quantitative aspects havenot yet been comprehensively addressed. This is a particularproblem regarding the fact that different compounds arelocated at different silica gel depths in dependence on theirRF-(retardation factor)-values. Therefore, IR-MALDI seems asuitable wayout because the IR radiation penetrates much dee-per into the silica gel than UV.

3. The achievable mass spectrometric resolution as well as massaccuracy is surely much lower if spectra are recorded directlyfrom a TLC plate in comparison to a standard metal MALDI tar-get. However, this is a minor problem in the field of lipid anal-ysis (in comparison to, e.g. tryptic digests of proteins) becausethe smallest mass difference within a certain lipid class thatmust be still resolved by MS is 2 amu, corresponding to onedouble bond.

5.1. Glyco- and sphingolipids

The majority of applications of TLC/MALDI have so far been ded-icated to the analysis of glycolipids, while phospholipids have beeninvestigated to a much lesser extent [22].

In a rather early attempt [191], native glycosphingolipids wereseparated on a conventional TLC plate and subsequently heat-transferred to a different membrane consisting of polyvinylidenedifluoride (PVDF) where the MALDI measurements were per-formed. This was done in order to remove impurities that arepotentially coming from the TLC material and because spectralquality and particularly the achievable sensitivity can be highlyimproved under these conditions.

In 2004, it could be shown that the analysis of gangliosides ispossible by direct TLC/MALDI without major fragmentations ofthe analyte [192]. In order to enhance the stability of the generatedions, these authors used a relatively high gas pressure to allow col-lisional ‘‘cooling” of the generated ions [192]. Recently is was alsoshown that glycolipids from brain can be analyzed by using a verysimple equipment, i.e. a commercially available MALDI-TOF MS de-vice equipped with a standard nitrogen laser [193]. The majority ofTLC/MALDI studies of glycolipids have used UV lasers. However,there are also some studies where infrared lasers were applied.These are primarily available on homebuilt MALDI devices butcommercially only on special request. Er:YAG (Erbium-doped yt-trium aluminum-Garret) IR lasers are normally used and havethe considerable advantage that IR radiation penetrates deeperinto the sample than UV radiation. Therefore, bringing the com-

plete analyte from the inner of the plate to the TLC surface is lessimportant. Beside an IR laser, Dreisewerd [194] and coworkersused an orthogonal but not an axial system for glycolipid analysis.The orthogonal configuration has the significant advantage thatirregularities of the surface of the sample do not reduce the achiev-able mass accuracy. It was found that even minor gangliosidesfrom a complex lipid mixture extracted from cultured Chinesehamster ovary cells can be identified.

These authors [194] provided also convincing evidence that thefluorescent dye ‘‘primuline” that is widely used in lipid research asit binds non-covalently to the fatty acyl residues [179] may be usedas a non-destructive, TLC/MALDI-compatible staining agent. Similardata were independently obtained by another group that addition-ally introduced ‘‘vibrational” cooling [195]. The term ‘‘vibrationalcooling” describes the desorption process in the pressure range,where ‘‘cooling” of the excess energy of the generated ions isachieved. Under these conditions, fragmentation of labile analytescan be minimized. This is normally performed by introducing an in-ert gas (e.g. N2) under a moderate pressure into the MS device.

In a recent study 2,5-dihydroxybenzoic acid (DHB) in acetoni-trile/water (1:1, v/v) was found very useful as matrix for the anal-ysis of glycosphingolipids because in this solvent mixture DHB ishighly soluble and this solvent mixture has a relatively high sur-face tension leading to reduced analyte spreading. Sensitivities be-tween 25 and 50 pmol could be obtained under these conditions. Itshould be noted that the application of highly concentrated matrixsolutions is very important because a larger excess of matrix overthe analyte in comparison to standard MALDI (i.e. the use of astainless steel target) is required for direct TLC/MALDI couplingin order to minimize fragmentation of the analyte [22].

A combination between MALDI, TLC and antibody detection wasalso recently suggested [196,197]: these authors used the follow-ing workflow: (a) TLC separation of cancer-associated glyco-sphingolipids (GSLs) from human hepatocellular and pancreatictumors (b) their detection with oligosaccharide specific proteinsand (c) the direct in situ MS analysis of the GSLs previously de-tected by the antibody. Detection limits of less than 1 ng of immu-nostained GSLs could be obtained. It is a particular advantage ofthis approach that crude lipid extracts of biological origin can bedirectly used for TLC-IR-MALDI-MS and no laborious, previousGSL purification is needed.

A ‘‘TLC-Blot-MALDI-Imaging” method was recently also sug-gested [198] to visualize individual molecular lipid species. Thismethod was indicated to have higher sensitivity than commonstaining methods and allows for direct visualization of all relevantlipids within a linear range of approximately one order of magni-tude. The important field of glycolipid analysis – particularly bycombined TLC/MALDI has been recently comprehensively re-viewed [199].

One important problem that is not only of relevance to TLC/MALDI but as well to MS imaging of biological tissues stems fromthe interference of matrix peaks with the signals of the moleculesof interest. In order to overcome this problem, it was suggested touse graphite-assisted laser desorption/ionization (GALDI) MS andthis technique has already been successfully applied to the analysisof cerebrosides in a total brain lipid extract of high complexity[200]. Beside the lack of typical ‘‘matrix” peaks it could also beshown that GALDI suffers far less from suppression effects, for in-stance, the suppression of cerebrosides by other dominant lipidspecies, particularly PC.

In addition to positive ion detection, (negatively charged) sulfa-tides could be also easily detected using a thin layer of graphite.Thus, graphite does not only enable the recording of positive butalso of negative ion spectra although details of the basic ionizationhave to be elucidated. So far, however, 9-AA seems to be the matrixof choice to record negative ion MALDI mass spectra [201].


5.2. Glycerophospholipids

Much less information is so far available regarding the directMALDI/TLC MS analysis of phospholipids: the direct analysis of lip-

Fig. 16. Expanded region of a TLC-separated egg yolk extract and the corresponding posthe plate. Only the relevant mass regions of each PL class are shown and assignments aretheoretical masses and were introduced to enable comparison with the experimental dadepending on the position where the laser hits the PE spot. The only marked fragmentpermission from Journal of Planar Chromatography 22 (2009) 35–42 [203].

ids separated on a TLC plate by MALDI-TOF MS was only recentlyattempted by two different methods. One approach is based onthe use of an IR laser and glycerol as matrix [150]. This approachhas the significant advantage that IR radiation penetrates deeper

itive ion MALDI-TOF mass spectra recorded directly from the indicated positions onprovided directly in the individual traces. Data given in parentheses correspond to

ta in selected cases. Please also note that the PE fraction provide different spectra,ation is the loss of the head group of SM (leading to m/z = 677.5). Reprinted with


into the TLC plate, but simultaneously confers the disadvantagethat abundant glycerol adducts (and to a minor extent even NaCladducts) of the PLs of interest are detected and complicate inter-pretation of the spectra. Another problem is that IR lasers are com-mercially barely available so far. Therefore, another approach useda readily available N2 laser and standard DHB as matrix [202].

One selected lane of a TLC-separated hen egg yolk extract andsome selected MALDI mass spectra are shown in Fig. 16.

Two facts are particularly remarkable in the context of Fig. 16.First, even rather minor fractions of, e.g. phosphatidylinositol (PI)can be easily analyzed and give spectra with a convincing signal-to-noise (S/N) ratio. As PI makes out only about 0.5% of the totalPLs of egg yolk [202], this clearly proves the significant dynamicrange of more than two orders of magnitude. Second, the lowerand the upper part of the PE spot provide significantly differentmass spectra, whereby PEs with longer fatty acyl (in particulararachidonoyl) residues are found in the upper and PEs with shorteracyl residues in the lower part of the spot. This is a clear indicationthat even the separation quality obtained under normal phase con-ditions is sufficient to allow the differentiation of fatty acyl resi-dues if MS is used for subsequent analysis [203].

Regarding detection limits, both approaches [150,202] providedcomparable results (about 400 pmol for PC detection) and, there-fore, both approaches might be useful for routine lipid analysis.This particularly holds as surprisingly good mass accuracies (about100 ppm) and mass resolutions (about 3000) could be obtainedthat are absolutely sufficient for the differentiation of individualPL species.

Very recently, an automatic routine for the spatially-resolvedscreening of a developed TLC plate became available and was al-ready successfully applied for the investigation of the lipid compo-sition of human mesenchymal stem cells [204] as well as plasmaPL signatures associated with respiratory disease severity in cystic

Fig. 17. Positive-ion MALDI-TOF mass spectrometric image (left) and densitometricimage (right) of a developed HPTLC plate containing lipids from hens’ egg yolk.Assignments of the individual spots and the m/z values of the signals that were usedfor image analysis are provided. Please note that two different spots may beobserved for most lipid classes and that lipids with ‘longer’ fatty acyl residues canbe easily differentiated form those with shorter fatty acyl residues. Reprinted withpermission from Journal of Planar Chromatography 22 (2009) 35–42 [203].

fibrosis patients [205]. This is a clear indication that TLC/MALDIdata can be basically quantitatively analyzed although further at-tempts are necessary to establish more useful and sensitive matrixcompounds – particularly for negative ion detection directly on theTLC plate [131,206].

Due to the much higher experimental time that is required torecord an MS image, 1D slices are normally investigated in the caseof a developed TLC plate. However, 2D MS images can be also easilyacquired and this is shown in Fig. 17.

MALDI MS imaging, a fast developing application of MS will bediscussed more comprehensively in the following section.

6. MALDI MS imaging

Microscopic methods and radiological investigation techniques(computer tomography, magnetic resonance imaging and sonogra-phy) are well-known imaging methods. These methods have beenestablished for several decades, whereas MS imaging is a rathernew, but very promising approach – not only in the basic sciencesbut as well in clinical diagnosis [207]. Although there are alreadyother MS imaging methods based on desorption electrospray(DESI) MS [208] or secondary ion mass spectrometry (SIMS)[209] available (for a timely review see [210]), we will focus hereexclusively to applications of MALDI MS. However it should benoted that DESI MS has clear advantages if the analysis of smallerlipids is of interest. For instance, it could be convincingly demon-strated upon the DESI image analysis of thin tissue sections of 68samples of human prostate cancer and normal tissues that choles-terol sulfate (m/z = 465) is a useful marker of prostate cancer [211].

Although common (commercially available) UV lasers penetrateonly a few lm into a solid sample such as thin tissue slices [20],such (for instance histological) slices can be directly analyzed byMALDI-TOF MS in order to obtain a two-dimensional concentrationprofile of the molecules in or at the surface of this tissue. The datasource for creating such images is a set of mass spectra acquiredstepwise with a certain spatial resolution and the intensities ofthe individual conventional mass spectra are afterwards trans-formed into a greyscale – the MS image. Although the majority ofMS suppliers offers nowadays complete imaging solutions, somespecial features should be taken into account:

1. The MS image is just a representation of spectral information.Therefore, the quality of the normal spectra determines thequality of the image and only such metabolites can be displayedthat provide a reasonable conventional mass spectrum. Thismeans that molecules are the easier detected the higher theirionization yields and the higher their concentration withinthe tissue slice. This surely makes lipids more easily detectablethan high mass proteins.

2. MS imaging is quite an expensive method. Handling hugeamounts of data in a very short time (laser frequencies of theorder of kHz are nowadays rather common) requires excellentcomputer hardware and data analysis software. Additionally,lasers with a high lifetime are required: imagine a sample of1 cm2. If this sample is scanned with a resolution of only100 lm, 10,000 individual mass spectra have to be recorded.If only 100 laser shots are averaged for each mass spectrum, thismeans that one million laser shots are necessary for a singleimage.

3. The achievable image quality is significantly dependent on thesample preparation and particularly on the even spotting ofthe tissue slice with the matrix. Already small inhomogeneitiesof the matrix deposition will seriously compromise the imagequality. Of course, this also holds for slight fluctuations of thelaser fluence.


Many of the points that have to be carefully considered in thecontext of successful MALDI MS imaging were recently summa-rized by Bernhard Spengler, one of the pioneers in this field [212]in [20]. Therefore, we do not want to give so many methodologicaldetails but will focus on some selected applications.

The simplest approach to perform MALDI MS imaging is to fixthe tissue slice onto the MALDI target and to cover it with matrix.The homogeneous spotting of the matrix is unequivocally the mostcritical point and many efforts for its improvements were and stillare undertaken [213] and devices for homogeneous matrix deposi-tion onto the sample of interest by sophisticated spraying tech-niques are nowadays commercially available.

Although not originally in the focus of interest, different classesof lipids are primarily detected if native tissue slices are investi-gated by MS imaging because these species ionize particularly welland lipids are abundant constituents of all tissues. Thus, a muchhigher interest in lipid analysis by MALDI-TOF MS has been obvi-ous over the last five years [214]. This particularly holds becausethere is increasing evidence that not only proteins but also certainlipid classes are useful as disease markers. For instance, it has re-cently been suggested that LPC may be used as a marker moleculeof ovarian cancer [215] and that differently saturated LPCs possesseither inflammatory or anti-inflammatory properties [216]. A typ-ical example of a positive ion MALDI image of a mouse brain isshown in Fig. 18

Fig. 18 shows the type of high quality images that can be gen-erated for individual lipid species in tissues using MALDI MS, inwhich clear anatomical distribution of these species are observed.This figure compares an optical image of a mouse brain sagittalsection with three 2D ion intensities maps generated by MALDIimaging. The MALDI images were recorded in positive-ion modeand sublimation of the matrix was employed to coat the tissue

Fig. 18. (a) Mouse brain, sagittal section, stained with Oil Red O. Section was taken 100during the tissue cutting process. (b) Mass spectrometric image of mouse brain sagittal seacquired with 50 lm plate movements, and is displayed smoothed, relative to the intensMass spectrometric image of mouse brain sagittal section acquired in positive-ion mmovements and is displayed smoothed with the scale intensity normalized to the mostsection acquired in positive-ion mode, displayed at m/z = 834.6. The image shown wasintensity normalized to the most abundant ion at m/z = 760.6. Reprinted with permissio

[213]. In addition to brain (that is often investigated because thistissue is particularly rich in a large variety of lipids [193]) the lipidcompositions of many different tissues have already been success-fully investigated by MALDI-TOF MS imaging [217] and thesecompositional data compared with results from established bio-chemical methods. A surprisingly good agreement between bothmethods is often found.

Therefore, tissue samples can be analyzed for their lipid compo-sitions by direct MALDI-TOF MS without sample workup [218].

It seems that the application of liquid ionic matrices providessuperior image qualities in comparison to conventional crystallinematrices due to the more even distribution of the matrix on the tis-sue surface [219]. This is the most important prerequisite for usingMALDI-TOF MS as a (quantitative) imaging technique [220], i.e. toobtain spatially-resolved information about the PL distributionwithin a given sample. Unfortunately, the achievable resolutionis normally not sufficient to obtain images with cellular resolutions[20]. Thus, the combination between MALDI and SIMS imaging is avery promising approach: while SIMS is capable of providingimages with a subcellular resolution, MALDI is unequivocally themethod of choice to detect lipids with relatively high masses suchas cardiolipins [221].

Although the lipid compositions of many different tissues (e.g.brain [218], eye lenses [220], and dystrophic muscle [222]) have al-ready been examined by MALDI-TOF MS, the most intriguing suc-cess of MALDI imaging was to prove that it is possible todifferentiate tumor tissue from the ischemic and necrotic areasof the lesion [223]. Already this selected example clearly indicatesthat MALDI imaging will have great future developments becauseit turns increasingly out to be useful for clinical diagnostics. A com-prehensive review dealing with MS imaging - not only of the re-lated lipid aspects - is available in [122].

lm lateral from the imaged slice. A tear in the tissue occurred in the pons regionction acquired in positive-ion mode, displayed at m/z = 760.6. The image shown wasity scale shown at the left of the image. The white bar indicates a 1 mm distance. (c)ode, displayed at m/z = 826.6. The image shown was acquired with 50 lm plateabundant ion at m/z = 760.6. (d) Mass spectrometric image of mouse brain sagittalacquired with 50 lm plate movements and is displayed smoothed with the scalen from J. Am. Soc. Mass Spectrom. 18 (2007) 1646–1652.


7. Summary

Unequivocally there is considerable interest in lipid analysisand it is expected that this interest will increase in the future be-cause many diseases are recognized to be accompanied by altera-tions in the lipid compositions of either body fluids or tissues oreven both. Thus, lipids are of unequivocal diagnostic relevance.Many different methods of lipid analysis are nowadays establishedand among these methods mass spectrometric techniquesunequivocally have the most significant potential [224]. The choiceof the most suitable method does not only depend on the suitabil-ity of the method to solve the problems of interest but also on theavailability of the different techniques. Since many MALDI massspectrometers were purchased in the context of proteomics andgenomics initiatives, it was one major aim of this review to provideevidence that these devices are not only useful in the field of polarbiomolecules but also for apolar ones, such as lipids: although notyet commonly accepted, MALDI-TOF MS represents a reliablemethod of lipid analysis. Hopefully, we were able to show in thisreview that basically all lipid classes can be analyzed by this meth-od - although some lipid classes are more refractive to analysisthan other ones. This particularly concerns relatively large lipids(cardiolipins), highly polar lipids (poly-phosphoinositides) and sul-fatides that tend to loose the sulfate residue. Here further method-ological developments - particularly in the field of (MALDI) matrixengineering - are required.

Nevertheless, MALDI MS has several important advantages:measurements can be performed in a short time and a very conve-nient way. Only minor, less time-consuming sample purification isrequired because considerable amounts of impurities such as saltsare tolerated. Therefore, MALDI-TOF MS is useful for the routineanalysis of a large number of samples. As soon as a suitable deviceis available, MALDI analyses may also be regarded as relativelyinexpensive in comparison to biochemical assays.

The interest in MALDI lipid analysis has been significantly in-creased with the establishment of MS imaging techniques duringthe past decade: as lipids are abundant in tissues due to the highnumber of cells and lipids ionize also particularly well, lipids are pri-marily detected under MS imaging conditions. As the spectral inten-sities are used to create the image, this is a strong indication thatquantitative analysis of lipid concentrations by MALDI-TOF MS ispossible. However, only certain lipid classes are detectable underthese conditions because individual phospholipid classes are char-acterized by different peak intensities: the concentration of com-pounds with quaternary ammonia groups such as PC or SM isoverestimated, whereas the concentration of other lipids is underes-timated. This holds at least if positive ion MALDI mass spectra are re-corded. Using additionally negative ion detection and/or different(for instance 9-AA and DHB) matrix compounds, this problem maybe at least partially overcome and it is assumed that different mark-ers of the different lipid classes will become detectable.

However, for the detailed analysis of complex mixtures, previousseparation of the sample into the individual lipid classes is required– in the same manner as if LC/MS would be used. For this purpose,combined TLC/MALDI may be used because the sample dispersedin a solid matrix (the silica gel) can be easily combined with a MALDIion source. Therefore, the same possibilities are available as in thecase of a LC/MS system. The future will show whether MALDI-TOFMS will become a method of similar importance as the so far estab-lished LC/MS methods in the field of lipid analysis.

Acknowledgments

The authors wish to thank all colleagues and friends whohelped them in writing this review. Particularly the kind and help-

ful advice of Dr. Suckau and Dr. Schürenberg (Bruker Daltonics,Bremen) as well as Dr. Thorsten Jaskolla (Goethe University, Frank-furt) is gratefully acknowledged.

This work was supported by the German Research Council (DFGSchi 476/12-1 and FU 771/1-1 as well as TR 67 project A2) and theFederal Ministry of Education and Research of the Federal Republicof Germany (‘‘The Virtual Liver”, 0315735).

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