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Progress in Lipid Research 49 (2010) 450–475

Contents lists available at ScienceDirect

Progress in Lipid Research


journal homepage: www.elsevier.com/locate/plipres

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: Although matrix-assisted laser desorption and ionization (MALDI) mass spectrometry (MS) – often but
Received 25 April 2010 not exclusively coupled with a time-of-flight (TOF) mass analyzer – is primarily established in the protein
Received in revised form 29 June 2010 field, there is increasing evidence that MALDI MS is also very useful in lipid research: MALDI MS is fast,
Accepted 1 July 2010
sensitive, tolerates sample impurities to a relatively high extent and provides very simple mass spectra
without major fragmentation of the analyte. Additionally, MALDI MS devices originally purchased for
‘‘proteomics” can be used also for lipids without the need of major system alterations.
Keywords:
After a short introduction into the method and the related ion-forming process, the MALDI mass spec-
MALDI-TOF MS
Lipids
trometric characteristics of the individual lipid (ranging from completely apolar hydrocarbons to com-
Phospholipids plex glycolipids with the focus on glycerophospholipids) classes will be discussed and the progress
Matrix achieved in the last years emphasized. Special attention will be paid to quantitative aspects of MALDI
MS because this is normally considered to be the ‘‘weak” point of the method, particularly if complex
lipid mixtures are to be analyzed. Although the detailed role of the matrix is not yet completely clear,
it will be also explicitly shown that the careful choice of the matrix is crucial in order to be able to detect
all compounds of interest.
Two rather recent developments will be highlighted: ‘‘Imaging” MS is nowadays widely established
and significant interest is paid in this context to the analysis of lipids because lipids ionize particularly
well and are, thus, more sensitively detectable in tissue slices than other biomolecules such as proteins.
It will also be shown that MALDI MS can be very easily combined with thin-layer chromatography (TLC)
allowing the spatially-resolved screening of the entire TLC plate and the detection of lipids with a higher
sensitivity than common staining protocols.
Ó 2010 Elsevier Ltd. All rights reserved.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
1.1. Soft ionization mass spectrometric methods. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 451
1.2. Fundamentals of MALDI mass spectrometry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 452
2. The role of the matrix – some practical considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 453
2.1. Typical MALDI matrices . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 454
2.2. Inorganic MALDI matrices. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455

Abbreviations: 9-AA, 9-aminoacridine; amu, atomar mass unit; APCI, atmospheric pressure chemical ionization; CE, cholesteryl ester; CHCA, a-cyano-4-hydroxycinnamic
acid; CID, collisionally-induced dissociation; DAG, diacylglycerols; DE, delayed extraction; DESI, desorption electrospray; DGDG, digalactosyl-diacylglycerol; DHB, 2,5-
dihydroxybenzoic acid; DIOS, desorption/ionization on silicon; DMAN, 1,8-bis-(dimethylamino)-naphthalene; EI, electron ionization; Er:YAG, erbium-doped yttrium
aluminium garnet; ESI, electrospray ionization; FAB, fast atom bombardment; FD, field desorption; FI, Field Ionization; FT, Fourier Transform; GALDI, graphite-assisted laser
desorption/ionization; GC, gas chromatography; GPL, glycerophospholipid; HPLC, high-performance liquid chromatography; HPA, hydroxy-picolinic acid; ILS, ionic liquids;
IP, ionization potential; IR, infrared; LD, laser desorption; LOD, level of detection; LOQ, level of quantification; LPA, lysophosphatidic acid; LPC, lyso(monoacyl)-
phosphatidylcholine; LPL, lyso(monoacyl)-phospholipid; MALDI, matrix-assisted laser desorption and ionization; MGDG, monogalactosyl-diacylglycerol; MS, mass
spectrometry; MTPFPP, meso-tetrakis(pentafluorophenyl)porphyrin; m/z, mass over charge; Nd:YAG, neodymium-doped yttrium aluminium garnet; NMR, nuclear magnetic
resonance; PA, phosphatidic acid; PC, phosphatidylcholine; PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, phosphatidylinositol; PIP2, phosphatidylinositol-
4,5-bisphosphate; PL, phospholipid; PLA2, phospholipase A2; PNA, para-nitroaniline; PPI, (poly-)phosphoinositides; PS, phosphatidylserine; PSD, post source decay; PVDF,
polyvinylidene difluoride; RF, retardation factor; ROS, reactive oxygen species; S1P, sphingosine-1-phosphate; SA, sinapic acid; SCX, silica gel cation exchanger; SIMS,
secondary ion MS; SM, sphingomyelin; S/N, signal to noise; sn, stereospecific numbering; SQDG, sulfoquinovosyl-diacylglycerol; TAG, triacylglycerol; TCA, trans-4-hydroxy-
3-methoxycinnamic acid; TFA, trifluoroacetic acid; THA, trihydroxy-acetophenone; TLC, thin-layer chromatography; TOF, time-of-flight; UV, ultraviolet.
* Corresponding author. Tel.: +49 341 97 15733; fax: +49 341 97 15709.
E-mail address: juergen.schiller@medizin.uni-leipzig.de (J. Schiller).

0163-7827/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.plipres.2010.07.001
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 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 455
3.2. Plant pigments. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
3.3. Flavonoids and carotenoids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
3.4. Free fatty acids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 456
3.5. Cholesterol and cholesteryl esters . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 457
3.6. Sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.7. Glycerolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.7.1. Di- and triacylglycerols . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 458
3.7.2. Glycoglycerolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.8. Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.8.1. Zwitterionic glycerophospholipids (PC and PE) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 459
3.8.2. Acidic glycerophospholipids. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 463
4. Analysis of lipid mixtures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 464
5. Separation of the individual lipid classes prior to analysis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 466
5.1. Glyco- and sphingolipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 467
5.2. Glycerophospholipids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 468
6. MALDI MS imaging . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 469
7. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471

1. Introduction nique less suitable for the analysis of mixtures. Therefore, the
prime application of EI MS in the context of lipids is nowadays nor-
We are currently living in an ‘‘omics” time period [1]. Although mally the analysis of free fatty acids that can be obtained by sapon-
this list is surely not complete, there were already proteomics, ification of lipids. This also holds for oxidation products derived
genomics, lipidomics, metabonomics, metagradomics, metallomics from free fatty acids and their metabolic products such as throm-
and – rather recently – interactomics. As these approaches provide boxanes or leukotrienes [7]. GC/MS is even nowadays a highly
an immense amount of data, bioinformatics is an indispensable established and widely used method for the quantitative analysis
counterpart leading to ‘‘systems biology” initiatives [2] that may of relatively apolar compounds. Unfortunately, sample preparation
help to get further insights into complex metabolic networks of is quite cumbersome and time-consuming. Because of these disad-
biological systems. vantages, the invention of soft-ionization methods, enabling the
As this review is dedicated to lipids and their analysis, a short analysis of molecules refractive to EI, must be regarded as a real
definition of ‘‘lipidomics” is necessary: according to a recently pro- milestone in the history of modern mass spectrometry [8].
vided definition [3], ‘‘lipidomics” can be defined as ‘‘the full charac-
terization of lipid molecular species and of their biological roles 1.1. Soft ionization mass spectrometric methods
with respect to expression of proteins involved in lipid metabolism
and function, including gene regulation”. The combination of all The differentiation between ‘‘hard” and ‘‘soft” ionization MS
these complex aspects is clearly a very challenging task, but the methods is a matter of philosophy and depends to a significant ex-
first step is obviously the qualitative and quantitative knowledge tent on the analyte of interest and its properties. However, all mod-
of the lipid composition of an unknown sample. ern ionization methods may be regarded as ‘‘softer” than EI [9].
Many ‘‘omics” applications are based on the application of mass Methods enabling the ionization of compounds refractive to elec-
spectrometry (MS) and would not have been possible without the tron collision ionization comprise gas phase ionization techniques
significant progress achieved in this field during the last decades. such as chemical ionization (CI) that was discovered in 1913 by
MS is an incredible powerful method and may be regarded as an Thompson [10], field desorption (FD) and field ionization (FI),
indispensable tool in physics, chemistry, biochemistry and methods based on particle bombardment such as fast atom bom-
(increasingly) medicine and particularly clinical diagnosis [4]. bardment (FAB) or secondary ion MS (SIMS) [11]. Each of these ion-
The history of MS began more than one century ago and was ini- ization methods has its individual strengths and weaknesses, but
tially related to the discovery of the different isotopes of the chem- the application of these methods is normally limited to special
ical elements [5] and, thus, primarily a task of physicists. For problems or selected analytes [4]. Only chemical ionization (often
instance, Thompson was awarded the Nobel Prize (of Physics) in in combination with atmospheric pressure, APCI) is used to a high-
1906 for his work on ‘‘Conduction of Electricity through Gases”. er extent for the analysis of lipids. For a recent review of applica-
In contrast, the history of the success of MS in biology and life tions of CI in lipid research see [12], while a survey of the
sciences is much shorter and directly related to the discovery of advantages and drawbacks of the different ionization techniques
new and gentle ionization methods: over many decades electron is available in [13].
ionization (EI) was exclusively available as ionization method [4]. Today, electrospray ionization (ESI) and matrix-assisted laser
EI makes use of fast electrons to ionize the analyte molecules in desorption and ionization (MALDI) MS are most often used and
the gas phase by the removal of one electron. Although this tech- the majority of commercially available MS devices is equipped
nique was (and still is) unequivocally suitable for the investigation with one (or maybe even both) of these ion sources. As a maximum
of small and/or volatile compounds, EI normally fails if larger mol- of information is obviously achievable if both techniques are com-
ecules with low volatilities are to be analyzed: although it could be bined, they should be regarded as complementary but not as com-
shown that the molecular ion (M ? M+ + e) of phospholipids is petitive. The importance of these both ionization methods was
basically detectable if EI is used for ionization of the sample, the obviously also the reason why the inventors of ESI and MALDI,
obtained results were not very convincing [6] and fragment ions i.e. Fenn and coworkers [14] and Tanaka and coworkers [15],
are much more abundant in comparison to M+ making this tech- 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- 1.2. Fundamentals of MALDI mass spectrometry
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 the A detailed survey of methodological aspects of MALDI MS is
related methodology. A review with a similar topic was already available in the excellent book by Franz Hillenkamp and Jasna
published by our group in 2004 in ‘‘Progress in Lipid Research” Peter-Katalinić [20]. Therefore, theoretical fundamentals of MALDI
[19]. However, many important improvements regarding potential MS will be only shortly discussed in this review. However, a short
applications, matrix optimization, quantitative data analysis and introduction is necessary for the less experienced reader.
more detailed characterization (particularly by MS/MS) of the MALDI MS is based on the utilization of a ‘‘matrix” that initially
different lipid classes have been achieved since that time and an absorbs the energy of the laser and mediates the generation of ions.
update of our previous review seemed necessary. In our opinion Although inorganic compounds such as graphite [24] or metal
the most important developments can be summarized as follows: oxide particles [25] may be also applied as matrix, small organic
molecules are used in the majority of cases and will be, thus, nearly
1. Although details of ion generation are still not yet completely exclusively discussed here.
understood, some progress in understanding this important Of course, the suitability of a certain compound as matrix is
aspect has been achieved. This is very important because fur- determined by the type of the laser and its emission wavelength.
ther theoretical insights will help to improve the performance Although many different lasers, including Nd:YAG (k = 355 or
of the MALDI method. A comprehensive survey of theoretical 266 nm) [26], excimer [27] or CO2 lasers [28] were already success-
aspects is available in [20]. fully applied, in the lipid field N2 lasers are primarily used.
2. Some new and improved matrix compounds were introduced Although IR lasers provide some advantages (see below), we will
that provide either higher sensitivities or higher reproducibili- focus here nearly exclusively on UV lasers with an emission wave-
ties than previously used matrices [21]. We will emphasize here length of 337 nm because the majority of commercially available
that some lipid-derived compounds are exclusively detectable if MALDI devices are equipped with this laser type. This is the reason
the most appropriate matrix is used. why the majority of the so far available lipid data were obtained
3. The coupling between MALDI MS and chromatographic separa- with UV lasers. A more comprehensive discussion of the role of
tion techniques is unequivocally a ‘‘hot” topic of current the matrix is available in [29].
research. This particularly concerns the combination between When the pulsed laser beam hits the sample (normally co-
MALDI MS and thin-layer chromatography (TLC) that helps to crystals of the matrix and the analyte), its energy is primarily ab-
overcome many problems related to mixture analysis in a sim- sorbed by the matrix that is present in a vast excess over the analyte
ple but elegant way [22]. (a 100–100,000-fold excess of the matrix is typically used). Conse-
4. MS imaging is nowadays an established method to monitor the quently, the matrix is vaporized, carrying intact analyte molecules
distribution of certain molecules within a tissue. As lipids ionize into the vapor phase. A simplified schema of the processes occurring
particularly well, they are very easily detectable under these in a typical MALDI-TOF mass spectrometer is shown in Fig. 2 [19].
conditions and this fact has pushed the interest in lipid analysis During the expanding process of this gas cloud, ions (e.g. H+ and
by MALDI MS considerably [23]. Na+) are exchanged between the matrix and the analyte, leading to
the formation of charged analyte molecules. These analyte ions are
The continuously increasing interest in MALDI MS as well as its called ‘‘adducts” or ‘‘quasimolecular ions” (sometimes the term
applications in lipid analysis is clearly reflected by the data given in ‘‘pseudomolecular” ions may be also found although the use of this
Fig. 1. term is discouraged by the IUPAC). Beside cation generation, an-
It must be explicitly stated that we do not want to suggest MAL- ions can also be generated by abstracting H+ or Na+ from the ana-
DI MS as the best method of lipid analysis. However, during the last lyte. The ratio between the cation and the anion yield is
centuries there were a lot of ‘‘omics” initiatives and many MALDI determined by the (gas phase) acidities of the analyte and the ma-
devices were purchased – particularly for protein and peptide trix [20] and fundamentals of the ion formation process were re-
analysis. It is our aim to convince the reader that these instruments cently comprehensively reviewed by Zenobi and Kochenmuss
are not exclusively useful in the proteomics field, but the same [30,31]. Recording positive-ion mode spectra is much more com-
instruments can be readily used for the analysis of lipids. There mon and it even seems (although not yet investigated in detail)
is surely no need to buy an additional device but the available that many MALDI mass spectrometers detect negative ions less
MALDI devices can be used without the need of major alterations! sensitively than positive ions.

Fig. 1. Number of scientific papers containing the term ‘‘MALDI” or ‘‘matrix-assisted” (a) and papers that contain additionally the term ‘‘lipid” or ‘‘phospholipid” (b). All data
were taken from the ‘‘Web of Science”™ database.
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 453

The most important difference between ‘‘molecular (radical) In the context of lipids, however, very high mass accuracies do
ions” (generated in conventional EI mass spectra) and ‘‘quasimo- not really provide a much larger extent of information because the
lecular ions” is the observed mass. As molecular ions are generated smallest mass difference within one given lipid class is 2 amu, i.e.
by the abstraction of one electron (the mass of which can be ne- one double bond. We will show below that there are some simple
glected for the majority of applications because it accounts for just chemical/biochemical methods (addition of certain salts or enzy-
about two-tenths of a percent of the mass of a proton) from the matic digest) to overcome problems related to potential signal
analyte, the mass of the generated ion (or the observed m/z ratio) overlap and a MALDI device with a high resolving power is not
corresponds to the mass of the analyte. In contrast, quasimolecular absolutely needed for routine lipid analysis.
cations are generated by the addition of a cation to the analyte. It should be noted that there is no absolute need to combine a
Therefore, the mass of the quasimolecular ion is characteristically MALDI ion source with a ‘‘TOF” mass analyzer. However, as MALDI
higher (normally 1 amu or 23 amu corresponding to the mass is used in the majority of cases for the detection of rather large
of H+ or Na+, respectively) in comparison to the analyte molecule molecules, the TOF detector is very popular because it has a nearly
[32]. unlimited mass range [20]. An additional reason is the pulsed (not
Despite its profound importance, the process of ion generation continuous) ion generation of MALDI that is most suitable for the
is so far only poorly understood and many papers are currently TOF mass analyzer. A more comprehensive survey of typical mass
dealing with this important topic. For instance, one recent issue analyzers as well as their individual advantages and drawbacks is
of European Journal of Mass Spectrometry (2006, Volume 12, Issue available in [13,20].
6) 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 actual 2. The role of the matrix – some practical considerations
measured quantity, the mass-to-charge ratio (m/z) may be re-
placed directly by the monoisotopic mass of the analyte molecule – Although there were already different attempts to predict the
plus or minus the mass of the ion required to generate a charge. suitability of an unknown chemical compound as MALDI (UV) ma-
The use of ‘‘Thompson” [Th] as unit of the mass over charge ratio trix by theoretical considerations, most matrices were (and actu-
was suggested nearly 20 years ago [34] but is not yet widely estab- ally are) found by accident or by a ‘‘try and error” approach. A
lished. A detailed discussion of this aspect is available in [35]. more detailed discussion of these aspects is beyond the scope of
After being formed, ions are accelerated in an electric field (typ- this review and we will focus exclusively on some selected practi-
ically of the order of 20 kV). After passing a charged grid, the ions are cal aspects. A ‘‘good” MALDI UV matrix in the practical sense is
drifting freely over a field-free space where mass separation is characterized by the following properties:
achieved: low mass ions arrive at the detector in a shorter time than
high mass ions [13]. This is the most simple, ‘‘linear” geometry of a 1. A suitable matrix should provide an excellent signal-to-noise (S/
TOF analyzer that is normally used for the analysis of larger mole- N) ratio for the peaks of the analyte of interest, i.e. a high sen-
cules because in this case high resolution is less important than high sitivity should be achievable.
sensitivity. Resolution and peak widths may be improved by using a 2. The matrix should provide a high absorbance at the emission
reflectron [20]. The reflectron enlarges the flight path and helps to wavelength of the laser. Thus, the required laser fluence should
compensate differences in the initial velocities of the ions during be as low as possible because enhanced laser fluence leads to
the ablation process. All spectra shown in this review were recorded enhanced analyte fragmentation and/or poorly resolved mass
on a MALDI-TOF device equipped with a reflectron. Using commer- spectra.
cial MALDI-TOF devices with reflectron configuration, mass resolu- 3. An optimum matrix is characterized by low background (i.e. the
tions of about 10,000 and mass accuracies higher than about 30 ppm signals of the matrix should be very small) in order to avoid
can be routinely achieved. This is rather poor in comparison to the interferences between the matrix and the analyte ions. It is
best available mass spectrometers nowadays which provide mass important to note that oligomers of the matrix are often gener-
accuracies lower than 1 ppm and also much higher resolutions. Such ated in the gas phase. Therefore, most matrices provide signals
high quality mass spectra are desirable in the context of proteomics, at m/z ratios much higher than their actual molecular weight!
where an increased mass accuracy helps to unequivocally assign un- 4. Finally, an ideal matrix should provide only a single adduct of
known (tryptic) peptides because the number of hits upon database the analyte and exhibit a weak tendency to cluster formation
searching is significantly reduced [36] if the mass can be provided because analyte–matrix clusters may seriously complicate data
with higher accuracy. analysis [37].

Reflector
Detector

mass to charge
(m/z)

Reflector
Matrix Nitrogen Laser ("electrostatic Mirror") Linear
Sample (337 nm) Charged
Plate Grid Detector

Sample Heavy IonsLi ght Ions


mass to charge
(m/z)
Target Field-free time-of-flight
High Voltage

Fig. 2. Schema of the processes occurring during the MALDI-TOF ionization process in the mass spectrometer (for details see text). The influence of the detection using the
‘‘linear” and ‘‘reflector” mode is emphasized in the figure. Reprinted with modification and permission from Elsevier [19].
454 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

As the vast majority of commercially available MALDI devices organic acids are acidic and, thus, capable of triggering the
are equipped with nitrogen lasers, the focus of this review will generation of H+ adducts. As sodium is an ubiquitous ele-
be on matrices compatible with N2 lasers (337 nm), whereas infra- ment, however, there are normally also Na+ adducts in addi-
red lasers and the corresponding matrices (such as glycerol or tion to H+ adducts. In addition to the enhanced generation of
succinic acid) will be treated only briefly. There are basically five H+ adducts, there is another practical reason to use carbox-
typical requirements that must be met by a powerful MALDI ylic acids: due to the presence of the aromatic ring system,
matrix and some selected UV MALDI matrix compounds are shown typical MALDI matrices are well soluble in organic solvents.
in Fig. 3. As MALDI is particularly used for the analysis of polar mole-
cules that are only scarcely soluble in organic solvents, water
(a) The matrix must exhibit a strong absorption at the laser is one important constituent of the most common solvent
emission wavelength, i.e. in the case of a UV laser normally systems: the presence of polar groups (such as carboxylic
at 337 nm. This is the reason why nearly all common organic acids) increases the solubility of the matrix in polar solvents
matrices contain an aromatic ring system with delocalized p [13].
electrons. Inorganic matrices such as graphite or metal salts (c) The matrix should be stable under high vacuum conditions.
will only be shortly discussed here because they are scarcely Although this sounds trivial, there are many potentially use-
applied to the field of lipid analysis so far [38]. As expected, ful MALDI matrices that do not – or not sufficiently – fulfill
the ionization efficiency increases if the absorption coeffi- this criterion. As most MALDI devices make use of high vac-
cient of the matrix at the laser wavelength increases and this uum conditions (normally about 1  109 bar) many com-
is one reason why among the different isomers of dihy- pounds tend to sublime. The loss of the matrix under high
droxybenzoic acids (DHB), only the 2,5 isomer that provides vacuum conditions may be one important reason why
the most marked absorption at 337 nm (but not the other MALDI mass spectra show time-dependent changes [40].
isomers) is commonly used as MALDI matrix [39]. Although (d) The matrix should isolate the generated ions and prevent the
the solution UV absorption of the matrix is determined in generation of analyte clusters, for instance, dimer formation.
many studies, it must be emphasized that exclusively the This is the prime reason why a significant excess of the
UV absorption in the solid state determines the absorption matrix over the analyte is normally required in order to
properties under MALDI conditions. Absorptions determined obtain optimum results. Additionally, the laser fluence
in the liquid and in the solid state may be significantly dif- should be kept as high as needed but as low as possible
ferent [39]. because matrix analyte clusters are increasingly generated
(b) The energy absorption and the subsequent evaporation of at elevated laser fluences [19].
the matrix must lead to the generation of ions from the ana- (e) The matrix and the analyte should give homogenous co-
lyte of interest. This is often believed to be the reason for the crystals. Improvement of homogeneity is clearly science of
use of carboxylic acids (many matrices are derived from cin- its own and depends – beside the analyte, the matrix and
namic or benzoic acids) as matrix compounds because the solvent system – on the method of sample preparation.
This important topic would be a review of its own and has
been reviewed elsewhere [41] but with the focus on polar
molecules. Briefly, the simpler the sample preparation
method, the lower is the homogeneity of the matrix/analyte
mixture. The most frequently used ‘‘dried droplet” method,
i.e. the successive deposition of matrix and analyte are nor-
mally not suitable if any quantitative data have to be derived
from the mass spectra. In contrast, the more sophisticated
sample preparation by electrospray deposition provides a
much more homogeneous matrix/analyte mixture and nor-
mally permits quantitative data evaluation. Although MALDI
targets made from different metals (e.g. aluminum, stainless
steel or gold) are nowadays commercially available, the
material of the target plays only a minor role under practical
laboratory conditions: using dried droplet preparations, the
sample/matrix layer is normally such thick that the laser
(that normally penetrates only a few lm into the sample)
irradiance does not depend on the composition of the target
material. A more detailed discussion of aspects of sample
preparations is available in [41].

2.1. Typical MALDI matrices

Among the hundreds of compounds that were suggested to be


useful as MALDI matrices, only a handful is used in daily practice
and many suggestions of matrices remained ‘‘a flash in the pan”.
As a complete survey of all matrix compounds (for a more detailed
review see the rather old but still very useful paper by Fitzgerald
[42]) would be beyond the scope of this paper, only the most
Fig. 3. Survey of some important UV MALDI matrices that are often used in the field
of lipid analysis. The molecule classes from which the individual matrices are
important matrices in the lipid field will be discussed in more de-
derived are also indicated. Please note that this is only a selection of the (in these tail in the subsequent paragraphs of this review. A more complete
authors opinion) most important matrices in the field of lipid analysis. survey is available in [29].
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 455

However, we would like to emphasize that lipids offer a consid- be found in living organisms. As we are sure that the readers of
erable advantage in comparison to polar molecules: as already ‘‘Progress in Lipid Research” will know the related chemical struc-
indicated above, the majority of MALDI devices make use of a UV tures, we will pay only minor attention to this point. Our previous
laser emitting at 337 nm. This normally requires matrix com- review in this journal [19] contained a survey of the related chem-
pounds with aromatic residues. Such compounds are only barely ical aspects and may be consulted if needed. Additionally, there
soluble in water, but well soluble in organic solvents. Thus, spectra was a recent paper that gave a comprehensive survey of the classi-
of apolar molecules can be recorded in a single organic phase with- fications of lipids [53].
out the need of adding water. This results in enhanced reproduc-
ibility and only relatively moderate shot-to-shot deviations [43] 3.1. Hydrocarbons and wax esters
that are often serious problems regarding the MALDI MS analysis
of polar molecules. As already indicated above, positive ion MALDI MS is much
more common than negative ion MALDI MS. In order to convert a
2.2. Inorganic MALDI matrices (neutral) molecule into a positively-charged ion, a cation, for in-
stance a proton must be added. Expectedly, this normally takes
As the matrix is normally applied in vast excess over the ana- place at sites with a high electron density, i.e. charged groups such
lyte, typical matrix peaks (often representing cluster ions with as phosphate or atoms with an electron lone pair such as nitrogen
higher masses than the original matrix compound) are normally or oxygen.
seen in the MALDI spectra – particularly in the smaller m/z range. Nevertheless, even completely apolar compounds (without any
This may lead to reduced sensitivities and the potential suppres- oxygen, nitrogen or sulfur heteroatoms) such as the unsaturated
sion of analyte signals. Therefore, the application of inorganic com- hydrocarbon squalene (the structure of which is shown in Fig. 4),
pounds that do not give any signals is a straightforward approach. a hydrocarbon containing several isoprene units, as well as its olig-
The very first application of an inorganic matrix was published omerization products can be easily analyzed by MALDI-TOF MS
by Tanaka and coworkers [15] who used glycerol suspensions of [54]. Squalene occurs in olive oil (about 0.8–12 g/kg) and has been
cobalt nanoparticles for the ionization of large molecules. The per- assumed to be related to the health benefits of vegetable oils and
formance of lm size metal and metal oxides such as Al, Mn, Mo, Si, is, thus, of significant nutritional interest.
Sn, SnO2, TiO2, W, WO3, Zn and ZnO for the detection of small or- Due to the apolarity of squalene, an auxiliary reagent such as
ganic molecules was also tested by Kinumi [25] using polyethylene silver trifluoroacetate (AgTFA) is often added to the matrix, for in-
glycol 200 and methyl stearate as selected ‘‘small” analytes. Higher stance, DHB [55] in order to improve the yield of ions.
sensitivities in comparison to classical organic matrices could be As many hydrocarbons contain olefinic residues and give, thus,
obtained and no interfering matrix background signals were de- rise to a significant UV absorption, simple laser desorption (LD) MS
tected. Generally, decreasing particle size results in increased sen- can also be used because an additional matrix is not absolutely
sitivity and, thus, nanoparticles instead of microparticles are necessary and does not significantly improve the spectral quality.
nowadays preferentially used. Although these metal or metal oxide This provides the significant advantage that there is no interfer-
particles do not give any ‘‘matrix” peak, they have a very high melt- ence with matrix peaks at all. However, direct LD analysis is impos-
ing point and, thus, enhanced laser fluences have to be used to sible if completely saturated hydrocarbons are to be analyzed as
transfer them into the gas phase. This often results in enhanced these compounds lack sufficient UV absorptions.
analyte fragmentation [44]. Another problem that hinders their Asphaltene fractions of crude petroleum [56] can be investi-
wider application is their limited commercial availability. gated in a similar way and also in this case the MALDI and the
Carbon nanotubes could be also successfully used and it has LDI spectra are of comparable quality. Additionally, applications
been shown that detection limits of certain analytes can be im- of MALDI-TOF MS to polyaromatic compounds [57] have also been
proved for one or two orders of magnitude [45]. An additional described although the insolubilities of these molecules conferred
advantage of these materials is that they can be used to enrich cer- some problems. These problems were solved by using a new sam-
tain apolar analytes due to their considerable hydrophobicity. Inor- ple preparation method consisting of mechanically mixing analyte
ganic materials such as silicon [46], graphite [47] and TiO2 [48] and matrix without the necessity to use solvents [58]. Finally,
have been used to coat the surface of the MALDI plate, and no addi- 7,7,8,8-tetracyanoquinomethane (TCNQ) was shown to possess
tional matrices were used. Fabrication of complete MALDI plates superior properties in comparison to other matrices [59]. A more
from porous silicon and graphite for matrix–less analyte detection comprehensive survey about LD MS is available in [60] and this re-
has also been reported [49]. Such inorganic oxides are especially views covers also aspects of desorption/ionization on silicon (DIOS)
useful because their isoelectric points vary over a wide range from as well as carbon-based microstructures. Finally, a quite old, but
acidic to basic. Therefore, the creation of positive or negative ions is nevertheless excellent review dealing with LD MS is available in
favored depending on the oxide choice. [61].
Some selected applications of these matrices will be described
at the appropriate places in this review. However, inorganic matri-
ces are not commonly used so far and there are only very few
applications to phospholipids available [50]. However, metal or
metal oxide nanoparticles seem particularly promising in the
imaging field because they give higher resolutions than standard
solid matrices [51]. Therefore, all these materials are assumed to
have significant future potential.

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 Fig. 4. Chemical structure of the physiologically important hydrocarbon ‘‘squa-
[52]. In this review we will deal exclusively with apolar com- lene”. Virgin olive oil is a rich source of this hydrocarbon and the squalene content
pounds and particularly with lipids and phospholipids that can of vegetable oils is often assumed to be important for the related health benefits.
456 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

It should be noted that the lithium salt of DHB is a more suitable only a minor extent of fragmentation is detectable if 20 ,40 ,60 -trihy-
matrix for hydrocarbon analysis than the free acid DHB: LiDHB droxyacetophenone (THA) is used as matrix, whereas DHB gives
provides less pronounced fragmentation and higher sensitivity much higher yields of fragment ions and is, thus, the matrix of
[62]. Similar data were also obtained in the case of wax esters choice to record post source decay (PSD) mass spectra [71]. It
[63] and insect cuticular hydrocarbons [64], whereby saturated was also reported in the context of red wine analysis that quanti-
and unsaturated hydrocarbons with more than 70 carbon atoms tative data can be directly achieved from the MALDI mass spectra
could be detected. In a very new paper it was shown that the dis- [72]. Due to the absorbance of these compounds in the UV range,
tribution of different wax esters and other apolar compounds can recording simple laser desorption spectra is a potential alternative.
also be investigated by MALDI imaging MS [65]. Of course, there It has been recently shown that direct imaging of plants is also pos-
are also many different apolar polymers the analysis of which sible [73]: as no matrix addition is required (that determines the
might be of significant interest. These compounds will not be dis- achievable resolution due to the size of the crystals), highly re-
cussed here but the interested reader is referred to the excellent solved (resolution about 10 lm) MS images can be obtained and
review by Batoy and coworkers [66]. it could be shown that the highly specific distribution of important
flavonoids such as kaempferol, quercetin and isorhamnetin can be
3.2. Plant pigments imaged at the cellular level. Similar data can be also obtained if col-
loidal graphite is used as matrix [74].
Since a pioneering MALDI work in 1996 [67], only very moder- In a very recent work it could be shown that flavonoids such as
ate efforts were undertaken to study chlorophylls and other plant quercetin or rutin may be used themselves as matrices for inor-
pigments [68]. This is somewhat surprising because these pig- ganic metal complexes [75] and provide better results in compar-
ments play a very central role in the plant metabolism, particularly ison to common crystalline matrices such as DHB.
regarding photosynthesis. Using standard DHB matrix, such mole-
cules are easily detectable. However, they are only detectable sub- 3.4. Free fatty acids
sequent to removal of the central metal ion [69]. Chlorophyll a, for
instance, is detected at m/z = 871.5, although its monoisotopic The MALDI-TOF MS analysis of free fatty acids is quite difficult if
mass is 892.5 (for chemical structure see Fig. 5). standard MALDI matrices are used. The most serious problem is the
The observed mass difference may be explained by the addition overlap of the signals of the free fatty acids with matrix signals that
of three H+ and the loss of Mg2+ leaving a single positively-charged are particularly abundant in the low mass range. Of course, this is a
ion. A very recent study [70] provided evidence that the loss of the serious problem if fatty acids at low concentrations have to be ana-
central ion can be avoided if terthiophene is used as the MALDI lyzed. According to our best knowledge standard matrices such as
matrix. In contrast, however, fragmentation of the phytol-ester DHB or CHCA (a-cyano-4-hydroxycinnamic acid) are not suitable
linkage was more pronounced in the presence of the terthiophene for this purpose, but some methods as to how this problem can
matrix. It seems likely that an enhanced laser fluence was needed be overcome have been suggested:
under these conditions due to the weaker UV absorption of this
matrix resulting in enhanced fragmentation. 1. If meso-tetrakis(pentafluorophenyl)porphyrin (MTPFPP), the
structure of which is shown in Fig. 6 [76], is used as matrix,
3.3. Flavonoids and carotenoids the problem of signal and matrix overlap can be overcome.
MTPFPP has a relatively high mass and does not give signals
These compounds can be easily characterized by MALDI-TOF below m/z ffi 500 in the positive-ion mode. Thus, fatty acids
MS although their tendency to give fragment ions seems strongly with typical masses between about 200 and 350 Da can be
dependent on the applied matrix. According to current knowledge, unequivocally detected. This was demonstrated, for instance,
with fatty acid mixtures obtained from different vegetable oils
subsequent to alkaline hydrolysis [77]. As an excess of sodium
acetate was added, exclusively the Na+ adducts of the Na+ salts
of the released fatty acids were detected. Therefore, there are no
problems with the overlap of different adducts, on the one
hand, and differences in fatty acyl compositions (the H+ adduct
of arachidonic acid (20:4) results in the same m/z ratio as the
Na+ adduct of oleic acid (18:1)), on the other hand, could be
completely avoided. This method was also successfully used
for the analysis of complex fatty acid mixtures from different
biological samples, for instance, rat plasma [78]. However,
MTPFPP is so far not widely used because this matrix gives rise
to unknown artifacts. Typical examples of positive ion mass
spectra of selected free (saturated (6a) as well as unsaturated
(6b)) fatty acids recorded in the presence of MTPFPP are shown
in Fig. 6. Although all saturated fatty acids are detected with the
expected m/z ratios, a mass shift of 14 amu to higher masses is
observed if unsaturated fatty acids are investigated [79]. For
instance, oleic acid is observed at m/z = 327.2 (Na+ adduct of
the sodium salt) as well as at m/z = 341.2. Although this effect
has not been carefully investigated yet, the above data might
indicate the oxidation of a methylene into a carbonyl group.
Additionally, the sensitivity of this MTPFPP matrix is rather
poor and at least lg quantities of fatty acids are required.
Fig. 5. Chemical structure of chlorophyll, a very important plant constituent that 2. Fatty acids are acidic compounds and should be, thus,
enables light fixation during the photosynthesis process. easily detectable as negative ions – at least if a sufficiently
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 457

alkaline matrix is used. 9-Aminoacridine (9-AA) seems a 3.5. Cholesterol and cholesteryl esters
particularly promising matrix in this field due to its consider-
able basicity (the pKa is about 9.99) [80]. Another advantage Although cholesterol is present in virtually all mammalian cells
of 9-AA is the very moderate background [81] provided by and body fluids [43] as well as in combination with significant
this matrix. It could be shown that the achievable sensitivity amounts of cholesteryl esters (CE) and triacylglycerols (TAG) in
is in the femtomolar range and linear detector response the lipoproteins of blood, only little interest has been paid to the
could be obtained over two orders of magnitude. Therefore, MALDI MS characterization of these molecules. One potential rea-
9-AA seems the matrix of choice for quantitative metabolomics son is the commercial availability of enzymatic test kits that help
studies of negatively charged compounds – not only fatty acids to determine both, cholesterol and cholesteryl ester concentrations
[82]. making MS methodology less important for the determination of
3. Very recently [83], it could be shown that 1,8-bis(dimethyl- these molecules. Using established MALDI matrices such as DHB,
amino)naphthalene (DMAN), a ‘‘superbasic” compound with a cholesterol is not detectable as the expected H+ adduct but only
pKa of 12.21 [84] is a powerful matrix because it enables the subsequent to water elimination at m/z = 369.3 (M+H+H2O)
detection of fatty acids (saturated and unsaturated ones) in [88]. Although it has been shown that the cholesterol concentra-
low picomole amounts. Therefore, both, 9-AA and DMAN seem tion in extracts of, e.g. human lipoproteins can be accurately deter-
more suitable than MTPFPP because all fatty acids can be easily mined by MALDI-TOF MS [89], the relatively small mass of
and accurately detected, while the MTPFPP porphyrin matrix is cholesterol confers problems: in the same manner as in the case
obviously less suitable for unsaturated fatty acids (cf. Fig. 6b) of free fatty acids, there is a considerable overlap between the cho-
due to the +14 amu artifact and the lower achievable sensitiv- lesterol peak and the matrix background. Of course, this is a partic-
ity. However, there are rumors that the use of DMAN may ularly important problem if diluted samples have to be analyzed.
decrease the sensitivity in the positive-ion mode significantly: To our best knowledge, there were so far no attempts to establish
residual amounts of DMAN may bind all available protons and a more suitable matrix for cholesterol analysis. In particular it has
reduce the achievable sensitivities by reducing the available not yet been clarified, whether 9-AA represents a useful alternative
amount of this cationizing species. matrix: 9-AA normally detects only charged lipids such as phos-
4. Free fatty acids are also detectable if inorganic matrices such as pholipids, while an additional cationizing reagent (e.g. sodium ace-
graphite [85] or porous silicon [86] (desorption/ionization on tate) is required to generate ions from molecules that do not
porous silicon (DIOS MS)) are used. Although the achievable possess charged functional groups. These aspects will be discussed
sensitivity is rather poor, deprotonated fatty acids could be eas- below in more detail and it will be outlined that the combination of
ily detected as negative ions. A more detailed survey of this different matrices represents the method of choice to obtain a
topic is available in [87]. complete data set [90].

Fig. 6. Positive ion MALDI-TOF mass spectra of mixtures of fatty acids using MTPFPP as matrix. In (a) a mixture of lauric (12:0), myristic (14:0) and palmitic acid (16:0) was
investigated, whereas in (b) a mixture of oleic (18:1), linoleic (18:2) and a-linolenic acid (18:3) was applied. Reprinted with permission and with slight modification from
Journal of Food Lipids, 9 (2002) 185–200 [79].
458 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

There have been surprisingly few attempts to investigate cho- 3.7.1. Di- and triacylglycerols
lesteryl esters by MALDI MS [89,91]: in the same way as observed Triacylglycerols (TAG) are important for the storage of energy
for di- and triacylglycerols (see below), CEs are exclusively detect- (fat tissues in living organisms), while diacylglycerols (DAG) that
able as adducts with alkali metal ions (particularly sodium), are normally generated from glycerophospholipids by cleavage of
whereas the H+ adducts are not detectable at all. Although not the polar headgroup under the influence of the enzyme phospholi-
yet carefully investigated, this might be caused by the same reason pase C are important lipid-derived second messengers [100]. The
as described for triacylglycerols [92]: H+ adducts of cholesteryl es- positive ion MALDI-TOF mass spectra of both, DAGs [101] as well
ters are less stable in comparison to the Na+ adducts and do not as TAGs [102] can be easily recorded with standard DHB. The
‘‘survive” the flight distance to the mass analyzer of the MALDI de- MALDI mass spectra give always exclusively the Na+ adducts,
vice without fragmentation. The experimental fact that the MALDI whereas H+ adducts are never detected, even if the solutions are
mass spectra of chromatographically pure CEs always exhibit a acidified [92]. Additionally, there are always intense fragment ions
small cholesterol peak is a strong confirmation of this mechanism. corresponding to the loss of one sodiated fatty acyl residue. Using
MALDI MS in combination with high energy collisionally-induced
dissociation (CID), the losses of these fatty acyl residues can be
3.6. Sphingolipids
used for structural elucidation of TAGs [103], i.e. the determination
which fatty acyl residue is located in which position. It is important
Sphingolipids and glycosphingolipids are currently a hot re-
to note that exclusively the Na+ adducts were useful for that pur-
search topic because these compounds may be regarded to be
pose while neither the K+ nor the Li+ adducts (that can be easily
indicative of ageing and as disease markers. As there are some
obtained if the spectra are recorded in the presence of the corre-
recent reviews dealing with the subject of ‘‘sphingolipidomics”
sponding alkali salts) gave indicative fragment ion spectra [103].
[93,94], we will deal here only rather shortly with these com-
The applied matrix has only a relatively weak impact on the
pounds but will discuss this topic in more detail in the context of
spectral quality, whereas strongly different sensitivities are
TLC/MALDI (see below). Here, we will focus primarily on the anal-
achieved in dependence on the used solvents. For instance, CHCA
ysis of the most abundant sphingolipid, sphingomyelin (SM), the
and DHB dissolved in a mixture of acetonitrile and water gave only
structure of which is shown in Fig. 7.
sensitivities in the pmol range, whereas DHB in acetone provided
SM is detectable in the same way as phospholipids and H+ as well
detection limits in the fmol range. Additionally, DHB provides a
as Na+ adducts occur. Very recently a combination between MALDI
weaker background than cinnamic acid derivates [104] that tend
and ESI MS was used to study changes of the sphingolipid composi-
to significant matrix cluster generation. This is even more impor-
tion of parasitic nematodes [95]. It could also be proven that changes
tant than the achievable sensitivities because TAGs (e.g. from veg-
of the lipid composition of tissues (and particularly of brain) often
etable oils) are normally available in huge amounts. In order to
affect much more the sphingolipids than the glycerophospholipids
have highly reproducible spectra, sample preparation is very
[96]. Additionally, SM is an important constituent of virtually all
important and particularly the ion content must be carefully con-
body fluids as well as tissues and, thus, particularly important in
trolled. For instance, it was recently shown that 9-AA detects
the context of imaging studies of tissues [97]. Surprisingly, in com-
TAG only in the presence but not in the absence of additional
parison to our previous review [19], only a few papers dealing with
cationizing reagents (‘‘dopants”) such as ammonium acetate [105].
the MALDI MS analysis of sphingolipids appeared. Nevertheless, it
It is somewhat surprising that independent of the used matrix,
became obvious that a large variety of sphingolipids can be quanti-
DAG and TAG appear exclusively as the sodiated molecules,
tatively analyzed by MALDI-TOF MS in the presence of an internal
whereas the H+ adducts are not detectable at all. A convincing
standard [98]: with sphingosylphosphorylcholine as the internal
explanation has been recently given [92] and is based on the obser-
standard, the relative peak heights of SM and ceramide monohexo-
vation that the generation of fragment ions can be significantly re-
side (CMH) could be used as a quantitative concentration measure
duced under alkaline conditions. Thus, it was suggested that the
and linearity could be obtained between 50 and 1500 ng SM as well
observed fragments actually arise from unseen protonated TAGs
as 5 and 150 ng CMH content, respectively. Nevertheless, despite
as their fragmentation occurs so rapidly and completely that pro-
the similarities of the headgroups, SM is less sensitively detectable
tonated TAGs are normally not observed. If the pH is increased,
in comparison to PC.
the H+ concentration is simultaneously decreased leading to re-
duced H+ adduct generation and, accordingly, to a lower yield of
3.7. Glycerolipids fragmentation products [92]. Very recently it has been shown that
the yield of TAG fragments can be significantly reduced if the MAL-
Triacylglycerols (where all hydroxyl groups of the glycerol are DI target is covered with a thin nitrocellulose film prior to deposi-
esterified with fatty acids) are very abundant in animal organisms, tion of the TAG of interest [106]. The nitrocellulose film
whereas compounds such as mono- or digalactosylglycerols are simultaneously improves the shot-to-shot and sample-to-sample
particularly abundant in plants [99]. We will focus here primarily reproducibility through the exhibition of a more homogeneous
on animals and only to a lesser extent on plant lipids. Therefore, matrix/analyte co-crystallization.
characteristics of TAGs (and to a minor extent diacylglycerols) will It has been repeatedly shown that MALDI MS is very suitable for
be primarily discussed. the 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 be
unequivocally assigned, bromination and hydrogenation was also
applied and combined with PSD fragment ion spectra [109].
Another important application of MALDI-TOF MS is the investi-
Fig. 7. Chemical structure of the backbone of sphingomyelin. ‘‘R” represents a gation of the changes occurring in oil samples during frying [111].
variable alkyl chain. This is a very important practical application because products
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 459

generated upon deep frying may include potentially hazardous membrane composed of considerable quantities of GPL and further
compounds [112]. Chromatographic enrichment of the polar com- apolar components such as cholesterol, lipids are easily detectable
pounds prevented mass spectrometric ion suppression, thus allow- in all MALDI imaging experiments of tissues [123].
ing the detection of minor species originating from thermal Despite the considerable technological progress that has been
oxidation. Particularly, diacylglycerols (DAG), oxidized TAG as well achieved regarding the available MS hardware [124], aspects of
as TAG dimers, and TAG fragments arising from the homolytic the ‘‘optimum” MALDI matrix (if it does anyway exist) have not
beta-scission of linoleyl, peroxy, and alkoxy radicals were found yet been clarified. Therefore, there is currently an increasing num-
to be indicative of the frying process. Recently, lipid compositional ber of papers dealing with ‘‘matrix engineering” [21,29].
data determined by MALDI-TOF MS and HPLC/APCI-MS were com-
pared [113]. It turned out that there were no major differences
making MALDI a suitable tool to investigate the lipid compositions 3.8.1. Zwitterionic glycerophospholipids (PC and PE)
of mixtures. Among all GPL so far investigated, phosphatidylcholine (PC) has
been by far, the most comprehensively studied. This has several
3.7.2. Glycoglycerolipids reasons:
Plant and algal photosynthetic membranes contain relatively
small amounts of phospholipids, but high amounts of three glyco- 1. PC is the most abundant constituent of cellular membranes – at
glycerolipids [99], monogalactosyl-diacylglycerol (MGDG), digalac- least as far animals or humans are concerned.
tosyl-diacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol 2. PCs are commercially available (for instance, from AVANTI Polar
(SQDG). Due to differences in biosynthetic pathways, these lipids Lipids, Alabaster, USA) with varying fatty acyl residues and,
contain high amounts of linoleic and, especially, alpha-linolenic thus, can be easily used as model compounds. Additionally,
acids which have to be taken in the diet of most animals. alkyl- and alkenyl ether derivates are also available.
All glycoglycerolipids of plants can be easily characterized by 3. PC possesses a quaternary ammonia group that contains a per-
MALDI-TOF MS and some selected mass spectra are shown in manent positive charge. Therefore, PC is most sensitively
Fig. 8. Please note that the characteristic mass difference of detectable in lipid mixtures – at least if the positive-ion mode
22 amu is not caused by H+/Na+ exchange, but both peaks corre- is considered.
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 of Regarding (3), i.e. the achievable sensitivity, it is important to
these compounds – in the same manner as TAG and DAG that they note that this is not only dependent on the MALDI device used
do never generate H+ adducts but exclusively Na+ adducts. and, thus, the available mass analyzer and detector but also signif-
Please note that the discussed glycoglycerolipids can be easily icantly on the applied matrix. Therefore, it is senseless to provide
separated by means of TLC [69]. Therefore, the relative contribu- detection limits without specifying the applied matrix. Since DHB
tions of the individual lipid classes can be most easily determined (2,5-dihydroxybenzoic acid) is one of the most established and
by TLC while the individual fatty acyl residues can be easily iden- best characterized matrix compounds [125], the majority of the
tified by MALDI-TOF MS. Finally, it has been shown by PSD MALDI available data were obtained with this matrix [126] and some se-
[114] that the fatty acyl residue in sn-1 position is preferentially lected data are given in Table 1.
lost. This preferential loss may also be used for the determination Table 1 clearly shows that different lipid classes exhibit very
of the positions of the individual fatty acyl residues. different detection limits and points out that the charge state of
It has also been shown that MALDI MS combined with TLC is a a lipid determines its detectability. Please note that sulfatides con-
suitable, generally applicable method of lipid analysis [22]. taining a negatively charged sulfate group are much less sensi-
tively detectable than other lipids in the positive-ion mode.
3.8. Glycerophospholipids Therefore, the comparison of the positive and the negative ion
mode is normally a straightforward approach and this is best done
Glycerophospholipids (GPL) are the most important subclass of by using different matrix compounds with different acidities, for
glycerolipids and merit, thus, a separate chapter. GPL are not only instance, DHB for positive ion and 9-AA for negative ion detection.
of in vivo relevance as important constituents of the cellular mem- This will be explained below in more detail.
branes, but some GPL (such as phosphatidic acid, phosphoinosi- The significant importance of the matrix on the quality of MAL-
tides and lysophospholipids) are also involved in signal DI mass spectra of phospholipids was already recognized in a pio-
transduction processes, i.e. they act as second messenger mole- neering study by Harvey [127]. Although a large variety of different
cules [115]. matrices were tested, sinapinic acid, a-cyano-4-hydroxycinnamic
Therefore, GPL are attracting increasing interest as potential acid and DHB gave the best results regarding the achievable sensi-
disease markers in important, socioeconomically relevant diseases tivity, resolution and the extent of (unwanted) fragmentation. It
such as atherosclerosis [116] or rheumatic diseases [117]. Many was also noted that individual PL classes are detected with extre-
different chromatographic, spectroscopic as well as MS methods mely different sensitivities [127]. Particularly PE is much less sen-
to assess the GPL composition of an unknown sample (e.g. an or- sitively detectable than PC in the positive ion detection mode
ganic extract of a body fluid) are known and have been reviewed (acidic PL such as PS are much more sensitively detectable as neg-
[118,119]. The schematic structures of some selected GPL that ative ions and will not be considered here): this is due to the per-
can normally be found in organic extracts from cells and body flu- manent positive charge of the quaternary ammonia group of the PC
ids and that will be primarily discussed below in this review are molecule, whereas the amino group of the PE can be easily depro-
shown in Fig. 9. tonated leaving the negative charge of the phosphate group [128].
Although ESI MS was so far primarily used in ‘‘lipidomics” stud- This is the reason why the positive ion spectra of cell or tissue ex-
ies [120], the number of lipid studies making use of MALDI MS is tracts are dominated by PC signals [121]. Therefore, separation into
increasing [121]. One potential reason for the nowadays extremely the individual lipid classes is normally needed in order to analyze
growing interest in lipid analysis by MALDI MS is coming from the detailed lipid composition of mixtures. In contrast, the pre-
‘‘MALDI MS imaging” that is currently experiencing significant ferred detectability of certain lipid classes (particularly PC, LPC
interest [122] and will be explained below in more detail. As all rel- and SM) may also be regarded as an advantage because the mass
evant tissues contain large numbers of cells and all cells possess a spectra are less complex [32]. The preferred detectability of PC in
460 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

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 lipids
represent 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 Lipids
150 (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 a
strongly 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 fatty
acyl 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.
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 461

comparison to PE is illustrated in Fig. 10 using an extract from hen AA (11f), while many different signals (primarily stemming from
egg yolk. the DHB matrix [104]) are observed in the presence of DHB
It is obvious from Fig. 10, that even if the PE is easily detectable (11e). Differences are still more obvious if the negative ion spectra
per se in the absence of PC, not even small PE peaks are detected if of POPC are compared: the signal at m/z = 744.6 in the negative ion
a total egg yolk extract is investigated without previous separation mode is caused by a loss of one methyl group from the PC (11h),
into the individual lipid classes. Therefore, mixture analysis should whereas only a cluster ion between DHB and POPC can be observed
be always regarded with great caution. in (11g) at m/z = 912.6 [132]. Although not yet investigated in de-
This is a particular problem in the case of PE because this PL is tail, it is evident that PC may be easily misinterpreted as PE (and
not (or only with very low sensitivity) detected as negative ion if vice versa) if negative ion spectra are recorded.
classical acidic matrices such as DHB are used [129]. Although the loss of a methyl group from PC is rather common
Using matrices such as para-nitroaniline (PNA) [130] or 9-ami- in the case of ESI MS, this mechanism was so far only described a
noacridine (9-AA) [105], this problem can be easily overcome. single time regarding MALDI MS [133]: Marto and coworkers have
However, it has been shown very recently that PC is also detectable shown that PC is (in addition to its detection as positive ion) also
in the presence of 9-AA as negative ion [131] subsequent to the detectable as negative ion if trans-4-hydroxy-3-methoxycinnamic
loss of a methyl group and this is exemplarily shown in Fig. 11. acid is used as matrix. In addition to the loss of a methyl group
Fig. 11 provides clear evidence that DHB and 9-AA result in [M-15], loss of a quaternary amine [M-60] and loss of the choline
characteristic differences. There are obviously only slight differ- headgroup [M-86] could be simultaneously detected.
ences between DHB and 9-AA in the positive-ion mode, although Although the discussion of the detectability of different lipids
the ratio between the H+ and Na+ adduct (e.g. m/z = 760.6 and either as positive or negative ions seems to be of academic rele-
782.6 in the case of POPC, 11a,b) depends on the matrix and 9- vance only, this is absolutely not true: being able to detect a PL
AA favors the generation of H+ adducts [105]. However, the nega- as negative ion, makes the determination of its fatty acyl composi-
tive ion mode spectra differ more significantly. For instance, a sin- tion much simpler. For instance, a certain PL containing one stea-
gle signal is obtained if POPE is investigated in the presence of 9- royl and one linoleoyl acyl residue, on the one hand, and two
oleoyl residues, on the other hand, result in exactly the same
masses. Using MS/MS, however, this problem can be easily solved
Table 1 by monitoring the loss of the individual fatty acyl residues. As fatty
Survey of relative detectabilities and the related detection limits of different
acids are more sensitively detectable as negative ions, it is much
important lipid classes. All data were recorded on a standard MALDI-TOF instrument
in the positive-ion mode using the reflector mode and DHB as matrix. Data were better to use the negative ion detection mode but this is only appli-
taken from [126]. cable if the parent ion gives a clearly detectable negative ion signal.
For a more detailed discussion of MALDI fragment ion spectra of
Lipid class Relative detectability (%) Detection limit (ng)
PLs see [114].
PC 100 0.020
Although DHB is so far most established, this compound is
PS 70 0.029
Sphingomyelin 70 0.029
surely not the most sensitive matrix: using clathrate nanostruc-
PG 40 0.050 tures (i.e. an inorganic matrix), it could be recently shown that
Galactocerebrosides 40 0.050 lysophosphatidylcholine (LPC) is already detectable if it is present
PI 20 0.100 in a concentration of about 7  1022 mol. As 1 mol is equivalent to
PE 2 1.000
6  1023 molecules this indicates that already about 10 molecules
Sulfatides 0.001 2000
of the analyte are sufficient to obtain a detectable signal [134].

Fig. 10. Positive ion MALDI-TOF mass spectra of an organic extract (obtained according to the method by Bligh and Dyer) of hen egg yolk. Trace (a) represents the mass
spectrum of the total extract, whereas trace (b) corresponds to the PC fraction and trace (c) to the PE fraction, respectively. All spectra were recorded with DHB as matrix.
Separation of lipids was performed by TLC prior to MS analysis. Although some further TLC fractions could be obtained, only the most relevant lipid classes are shown here.
The dotted lines indicate the peaks of the most abundant PE species, PE 16:0/20:4.
462 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

744.6
912.6

681 (*) O CH3

754.6 O P O CH 2 CH2 N CH3


673.4 699.4 O CH3
821.5
(g) (h)

681 (*) 716.5

675 (*) O H
857 (*)
716.5 892.5 O P O CH2 CH 2 N H
O H
752.5 782.5
(e) (f)
740.5 740.5

762.5
O H

718.5 762.5 O P O CH2 CH 2 N H


577.5 603.5 585.0 O H
706
(c) 630.5 (d)
760.6 760.6
COOH NH2
O CH3
OH 782.6
O P O CH 2 CH2 N CH3
O CH3
650.5
HO N 782.6
(a) 672.5 (b)

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

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 the
sample 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. The
polarities of the performed measurements and the headgroups of the investigated lipid species are indicated in the figure. Reprinted with modification and permission from
Anal. Bioanal. Chem. 395 (2009) 2479–2487.

Although this approach is surely not broadly applicable in practice, the mass spectrometric detection of oxidatively modified lipids is
this low detection limit sheds light on the high sensitivities of available in [142].
modern MS devices and particularly their detectors. Unfortunately, MALDI-TOF MS investigation of the products of
The high sensitivity for detecting PC is obviously an advantage if the reaction between HOCl and PE is much more difficult: PE con-
derivatives of PC (i.e. with the same headgroup) are of interest. This tains, in comparison to PC, an additional reactive functional group,
particularly concerns LPC that lacks in comparison to PC one fatty the amino group that exhibits a higher reactivity in comparison to
acyl residue – normally in sn-2 position [135] – and the PC/LPC ra- the olefinic residues. Therefore, the prime reaction products are
tio has been proven to be useful as disease marker for instance of mono- as well as dichloroamines. It was shown that these products
rheumatic diseases [117]. It has also been shown that LPC is not can be detected by electrospray MS but not by MALDI-TOF MS be-
only generated by the (most widely established) enzyme phospho- cause decomposition occurs rapidly [143]. However, this only
lipase A2 (PLA2) but as well under the influence of reactive oxygen holds if standard matrices such as DHB or CHCA are used. In con-
species (ROS) such as HOCl [136]: in the first step of the reaction, trast, the expected products are easily detectable if a chlorinated
HOCl is added to one (or several) double bonds of the PC to gener- cinnamic acid (4-chloro-a-cyanocinnamic acid) derivate is used
ate the corresponding chlorohydrins. The characteristic mass shift [144] and the corresponding positive ion MALDI mass spectra are
of 52 amu can be easily monitored by MS. The introduction of these shown in Fig. 12.
strongly electronegative atoms (chlorine and oxygen) leads to the The underlying mechanisms for these surprising difference
reduction of the strength of the ester bond making hydrolysis of were discussed in detail and related to the different proton affini-
this bond easier and finally to the generation of LPC [137]. Thus, ties of the individual matrices in [145]. This example proves clearly
LPC is obviously a marker of inflammatory conditions accompanied that matrix design in order to develop more suitable matrices is
by oxidative stress. Of course, in addition to HOCl there are many very important and not only of academic interest.
more ROS that are generated under inflammatory conditions lead- Finally, in addition to diacyl PLs there are also lipids with alke-
ing to different lipid oxidation products [138]. nyl ether bonds and these compounds are commonly termed
Although oxidation products such as endo- or hydro-peroxides ‘‘plasmalogens” [146]. Although these compounds can be analyzed
are also basically detectable by MALDI-TOF MS, they are only de- in exactly the same manner as common PCs, there is one important
tected with much lower intensities [139,140]. Even if the detailed difference: plasmalogens are extremely sensitive towards even
reasons are not yet known, it seems reasonable to assume that la- traces of acids that leads to the cleavage of the alkenyl ether link-
ser irradiation leads (at least) to partial degradation of these ini- age under generation of the corresponding LPC. Although the acid-
tially formed products under generation of carbonyl compounds ity of MALDI matrices such as DHB is tolerated, the use of TFA
upon cleavage of the original double bond. Very recently 6-aza- (trifluoroacetic acid) as an cationizing agent is strongly discour-
2-thiothymine was suggested as the matrix of choice to detect oxi- aged because TFA in a concentration of about 0.1% is already suffi-
dized phospholipids [141]. A more detailed review dealing with cient to induce partial hydrolysis of plasmalogens [147]. This is a
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 463

714.5

782.5
784.5
736.5 804.5 806.5
692.5
(c)

736.5

714.5
(b)

714.5

736.5

692.5

(a)

680 700 720 740 760 780 800 820


m/z[Th]

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 were
recorded 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 804.5
corresponding 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 of MALDI-TOF MS subsequent to the removal of interfering PC by
interest because these are characterized by high plasmalogen con- its binding to a small column filled with a silica gel cation exchan-
tents [146]. ger (SCX). A more comprehensive review of this important topic is
available in [152].
3.8.2. Acidic glycerophospholipids Although PS is a lipid that is of high diagnostic relevance partic-
As expected, phospholipids such as PI, PS and PA are detectable ularly in the context of assessment of apoptotic cells [153], surpris-
with rather poor sensitivities as positive ions due to their negative ingly few attempts have been made to characterize PS by MALDI-
charge [126]. Additionally, these lipids occur normally in rather TOF MS. To our best knowledge there are just a very few papers
small amounts and, thus, they are rather difficult to detect. As they dealing with this topic: MALDI-TOF MS was recently used to inves-
are not commercially available to such an extent as PC and PE and tigate the lipid composition of tear samples from normal and dry
are often much more expensive, they have been much less often eyes of rabbits [154], whereby a solid ionic crystal MALDI matrix
investigated although they often have higher biological relevance of para-nitroaniline and butyric acid was used that gave superior
than the bulk phospholipids, PC and PE. results in comparison to common crystalline matrices. In addition
This particularly concerns phosphorylated phosphoinositides to an enhanced SM content in the dry eye tears, PS species were
(PPI) that possess three or more negative charges due to their addi- readily only detectable in dry eye tears but not in the normal tears.
tional phosphate residues [148]: all phosphoinositides produce Using artificial PL membranes, the influence of HOCl on PS was
excellent signals in the negative ion mode when analyzed as iso- investigated and many different products were detectable. As al-
lated compounds in amounts of about 1 nmol. However, the detec- ready discussed above, chlorinated compounds are only detectable
tion thresholds for phosphoinositides are higher than that of more if 4-chloro-a-cyano-cinnamic acid is used as a matrix [155] but not
abundant PLs and increase further with the phosphorylation state. in the presence of standard matrices.
While PC, for instance, is detectable in amounts of less than one The detection of phosphatidic acid (PA) and derived lyso-phos-
pmol, the amount of PIP3 on the sample plate that is needed to pro- phatidic acid (LPA) is a quite challenging task for the same reasons
duce a sufficient signal is higher than 700 pmol [149]. Similar data as the phosphoinositides: PA and LPA possess a high negative
were also reported regarding the detection of cardiolipin [150] that charge density and are, thus, much less sensitively detectable than
is also more highly charged than common PLs. Beside the charge, it common zwitterionic phospholipids. Additionally, PA and LPA are
might also be possible that the increased polarities of these com- only present in very small amounts in biological samples. In order
pounds lead to reduced interactions with the matrix that is crucial to overcome these problems, an inorganic zinc complex ([1,3-
for the ionization process. However, comprehensive investigations bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato] dizinc(II)) was
why phosphoinositides and cardiolipins are only scarcely detect- used as matrix by Tanaka and coworkers [156] in order to trap
able by MALDI MS are not available so far. This is a pity because the LPA and to significantly enhance its detectability. 0.1 pmol of
these compounds are particularly important, functional molecules. LPA 18:1 was detectable on the MALDI target under these condi-
Recently it has been shown [151] that PI species can be easily tions. This method was very recently improved and extended to
monitored and even quantified in rather complex mixtures by sphingosine-1-phosphate (S1P) in order to make this approach
464 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

applicable to high throughput screening of samples with signifi- According to our experience there is no absolute need to use an
cant clinical interest [157]. isotopically labeled reference compound, but a lipid with odd
numbered fatty acyl lengths may also be used. So far, there is no
agreement that a special standard must be used for each lipid class,
4. Analysis of lipid mixtures or if a single compound (each for positive and negative ion detec-
tion) is sufficient. Although relative peak intensity ratios may also
The different detection limits of the individual PL classes as de- be used, this method does not allow one to judge if both or only
scribed above and exemplarily reviewed in [126] are extremely one concentration are changing and, thus, the use of internal stan-
important for the analysis of biological samples: if crude organic ex- dards is recommended.
tracts from cell cultures, body fluids or tissues are to be investigated It is also highly recommended that the positive ion spectra are
by MALDI-TOF MS without previous chromatographic purification, recorded in the presence of an acidic (e.g. DHB) and the negative
it may happen – depending on the composition of the sample – that ion spectra in the presence of an alkaline (e.g. 9-AA) matrix. If
only some selected lipid classes, particularly PL with quaternary DHB, the most common matrix in lipid analysis is used, PC and
ammonia groups are detected while the remaining ones are sup- other PLs with quaternary ammonia groups such as LPC or SM,
pressed [158]. It is also a matter of fact that the different lipid classes are detectable as positive ions, whereas negatively charged PLs
influence the intensities of others and it is often observed that such as PI and PS are only detectable as negative ions or just
MALDI mass spectra change if they are recorded at different analyte with very low sensitivity in the positive ion detection mode.
concentrations. This is illustrated with an extract from egg yolk as a Using careful sample preparation, quantitative data regarding
typical example (Fig. 13): it is evident that spectra change signifi- the compositions of different cells [105], lipoproteins [89] as well
cantly with increasing dilution and particularly the apolar compo- as meat [162] could be obtained. These are only some selected
nents, i.e. the triacylglycerols (TAG) are only detectable if diluted examples but MALDI MS can surely be used to obtain quantita-
solutions are investigated. Such suppression effects are well known tive data. A more comprehensive survey of quantitative aspects
in MALDI-TOF MS and were thoroughly studied regarding the sup- of MALDI MS regarding small molecule analysis is available in
pression of matrix signals [159] as well as analyte signals [160]. [163].
Please also note that the ratio between the H+ (m/z = 760.6) and There is another important aspect beside mass spectrometric
the Na+ (m/z = 782.6) adduct of PC 16:0/18:1 is not constant but measurements: the influence of extraction on the lipid yield. Mix-
the Na+ adduct is obviously favored with increasing dilution. A more tures consisting of chloroform and methanol are the most estab-
detailed discussion of these effects is beyond the scope of this paper, lished and used in the majority of cases [118]. Although this
but can be summarized as follows: when more analyte is present topic would be worth its own review, the following critical aspects
than primary ions, suppression occurs as a consequence of second- should be considered:
ary reactions and this effect is also significantly dependent on the
applied laser fluence. It is well known to each MALDI user that there 1. Enrichment of lipids by extraction with chloroform (according
is a direct relation between signal intensity and analyte concentra- to the methods developed by Bligh and Dyer [164] or Folch
tion only in a small concentration range, while the achievable signal et al. [165]) works quite well for bulk lipids such as PC or PE
intensity decreases at higher concentrations. If the applied amount and these lipids are nearly quantitatively extracted. However,
of sample exceeds a certain limit, no signal is detectable anymore. more polar compounds such as LPA or PPI are incompletely
This behavior has been recently explicitly described [161]. There- extracted because these compounds have a higher affinity to
fore, it is highly recommended to use similarly concentrated solu- remain in the water/methanol phase. If these molecules are of
tions of the analytes and to perform all measurements in the prime interest more polar solvent mixtures and/or acidifying
presence of an internal standard. the solution is required.

Fig. 13. Positive ion MALDI-TOF mass spectra of an organic extract from hen egg yolk depending on the amount of sample. All samples were mixed 1:1 with matrix solution
prior to deposition onto the MALDI target. The egg yolk extract was diluted with CHCl3 in several steps as indicated in the figure.
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 465

2. Surprisingly, apolar lipids are also incompletely extracted if 20 preparation is shown: although we do not want to discuss this
standard methods such as the Bligh and Dyer approach [164] spectrum in detail (further details are available in [167,168]), it is
are used and more apolar conditions should be used for that evident that the spectrum of this detergent is very complex and
purpose. The reason for this incomplete extraction is most covers a significant mass range. This makes detergent removal
probably the tendency of apolar lipids to form other structures rather difficult and, therefore, the application of detergents to ex-
than amphiphilic PL that provide well-defined supramolecular tract lipids from biological sources seems not to be the method
structures. of choice. Nevertheless, there are some papers dealing with this ap-
3. If an aqueous sample containing proteins is treated with proach and the use of dodecyl maltoside for membrane solubiliza-
organic solvents, precipitation of proteins and other highly tion seems preferential because this detergent exhibits a lower
polar compounds will occur. This is obvious by the generation extent of heterogeneity [169]. The reader should also note that
of a white ring at the interface between the aqueous and the parameters such as the salt concentration influence the achievable
organic phase. Some lipids may stick to the precipitated pro- spectral quality although this is a much smaller problem in the
teins and are, thus, lost if only the organic layer is used for fur- case of lipids because the majority of the salt is already removed
ther analysis. by the extraction process [170]. Nevertheless, in order to account
for different salt concentrations, all adducts (normally the H+ and
In order to make a long story short: there is obviously not a sin- Na+ adduct) of a selected lipid must be used for subsequent data
gle method that allows the unequivocal extraction of all PL classes analysis. This means that the combined intensities of both adducts
and great care is required if absolute, quantitative data are re- must be used for proper quantitative analysis.
quired. There was one 31P NMR based study that has compared Regarding mixture analysis, one should also wonder if complete
the use of detergents and organic solvents for the (lipid) extraction information is really always needed. Would less sometimes be
of human erythrocytes [166]. It was one result of this study that more? This particularly holds because the difficulties of proper
extraction with the detergent sodium cholate in comparison to or- data analysis often strongly increase as more data are collected.
ganic solvents resulted in a much higher yield of PLs and would, According to our opinion it is often sufficient to have some defined
thus, be the method of choice in order to extract lipids quantita- selected metabolites determined. For instance, we have established
tively. Unfortunately, this method is only applicable to 31P NMR the PC/LPC ratio as a measure of the quality of a human ejaculate
where the presence of the detergents does not play a role because [171] that correlates very well with the amount of annexin-V-po-
they are not detected at all. In contrast, interference has been sitive spermatozoa. Very recently, we have also shown that this
shown recently following the treatment of boar spermatozoa with is not only possible for human spermatozoa but may also be ap-
different detergents and subsequently assessing the lipid composi- plied to the analysis of animal spermatozoa, for instance from cats
tions of the extracts by MALDI-TOF MS: in the presence of even and cattle [172]. The structures of the related molecules are shown
small amounts of detergents, the sensitivities for lipid detection in Fig. 15. Although it is not yet clear whether (a) increased activity
were strongly reduced [167]. Thus, the potential presence of deter- of PLA2 or (b) an enhanced generation of ROS or (c) a reduced
gents is a considerable problem: detergent removal kits are avail- reacylation of the LPL primarily contribute to the changed PC/LPC
able for proteins, but there is, so far, no commercial detergent ratio, this ratio seems a suitable measure of the quality of sperma-
removal kit for the organic (lipid containing) phase. This is caused tozoa. A very simple method to quantitatively extract LPC from
(a) by the lower interest that lipids were so far experiencing and aqueous samples that uses only methanol and necessitates only a
(b) the fact that most detergents exhibit a significant distribution single centrifugation step was suggested recently [173].
of their molecular weights. This makes their removal much more Somewhat related to spermatozoa research, it was very recently
difficult in comparison to compounds with a defined molecular shown that different animal oocytes provide very different MALDI
weight. In order to illustrate this fact in Fig. 14 the positive ion mass spectra and can be, thus, easily differentiated [174] and give
MALDI-TOF mass spectrum of a commercially available TWEEN molecular fingerprints of the lipid composition.

Fig. 14. Positive ion MALDI mass spectrum of commercially available TWEEN 20, a common detergent for the solubilization of cellular lipids. The spectrum was recorded with
DHB as matrix. Please note the considerable distribution of the masses due to variable numbers of bismethylene oxy groups. Please also note that there are at least two
different groups of peaks that are stemming from different structures. These are shown in the upper part of the figure.
466 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

5. Separation of the individual lipid classes prior to analysis moved by changing the polarity of the solvents. Afterwards the
silica gel with the attached lipid is scraped off from the TLC plate,
When separated, all phospholipid classes (if present in suffi- the lipid is eluted by organic solvents and subsequently analyzed
cient amounts) are easily detectable by MALDI MS because sup- by MALDI MS. Some selected examples of this technique are the
pression effects between the individual lipid species do not play analysis of characteristic lipid oxidation products [180], cells such
any role. Nevertheless, detection limits for the individual lipid clas- as bull [147] or human spermatozoa [181], body fluids such as lung
ses [126] are very different due to the reasons discussed above. surfactant [182], tissues such as brain [183] or organic extracts
Separation into the different lipid classes may be of course per- from algae [69].
formed by offline HPLC and the fractions obtained subsequently Although this approach is capable of providing convincing re-
analyzed by MALDI-TOF MS. We will not pay major attention to sults, it is obviously boring and time-consuming, particularly if
this application because HPLC is normally not coupled to a MALDI many samples each containing a lot of different lipid species have
but to an electrospray ion source and this important method of li- to be analyzed. Additionally, this method bears the risk of losing
pid analysis has been already comprehensively reviewed recently certain lipid species. For instance, it has been shown that an ‘‘effect
[175]. This also holds for the use of, for instance, APCI instead of of chromatography” exists [184]. This means that lipids with dif-
ESI [176]. ferent fatty acyl residues are differently tightly bound to the silica
In contrast, we will focus here on thin-layer chromatography gel and are, thus, eluted to a different extent. Therefore, the fatty
(TLC), that is even nowadays still a widely used separation method acyl compositions monitored prior and subsequent to chromatog-
in lipid research [177] due to its simple and fast (several samples raphy may be different [184].
can be separated at the same time) performance as well as the very Despite these problems, a method enabling a more direct cou-
moderate price of the required equipment. TLC is also very ecolog- pling between TLC and ESI MS was recently successfully estab-
ically-friendly because much lower amounts of solvents are lished [185]. A plunger based extraction interface (now
needed in comparison to HPLC. Finally, certification of TLC separa- commercially available as the ‘‘ChromXtract” from the CAMAG
tions is simpler in comparison to HPLC: using TLC, a completely company) combined with an HPLC pump was shown to provide
new stationary phase is always used, whereby potential contami- good results for quantitative TLC/ESI MS from HPTLC silica gel
nations from a previous run can be surely avoided, while in the plates regarding repeatability of the MS spectra and the achievable
context of HPLC one may always suspect that there is some resid- limit of detection (LOD) and the limit of quantification (LOQ). This
ual amount of the analyte even subsequent to intense washing of was shown using harmane, a heterocyclic aromatic amine, as the
the column [178]. selected model compound [186]. Modifications of the device
Clearly, TLC separation may be also offline coupled to MALDI: enabled complete, highly reproducible extraction of analytes
the lipid mixture of interest is separated by TLC and afterwards from glass backed as well as aluminum backed TLC and HPTLC
stained by a non-destructive dye that does not lead to alterations plates, layers with thicknesses up to 100 lm and different station-
of the masses of the lipids of interest. This requirement is fulfilled, ary phases [187]. This application is surely a current ‘‘hot” topic
for instance, by the fluorescent dye primuline that binds non-cova- [188,189] and of interest for scientists working in different
lently to the fatty acyl residues of lipids [179] and can be easily re- fields.

Fig. 15. Survey of the conversion of PC into LPC under the influence of the enzyme PLA2 or ROS. Please note that human spermatozoa are particularly rich in PC 16:0/22:6,
while a high plasmalogen content is characteristic of many animal spermatozoa. Accordingly, different products may be expected because the acyl- and the alkenyl-linkage
exhibit different stabilities.
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 467

Since a ‘‘solid” sample is used in TLC, i.e. the lipid is dispersed in plete analyte from the inner of the plate to the TLC surface is less
a solid silica gel ‘‘matrix”, the idea to combine MALDI directly with important. Beside an IR laser, Dreisewerd [194] and coworkers
TLC seems a straightforward approach and was already attempted used an orthogonal but not an axial system for glycolipid analysis.
at the end of the 1990s [190]. MALDI characterization of the devel- The orthogonal configuration has the significant advantage that
oped TLC plate would offer the advantage of much higher resolu- irregularities of the surface of the sample do not reduce the achiev-
tion because the achievable resolution is determined primarily able mass accuracy. It was found that even minor gangliosides
by the laser spot size that is normally of the order of only about from a complex lipid mixture extracted from cultured Chinese
20 lm. This means that 50 individual MALDI mass spectra can be hamster ovary cells can be identified.
recorded from a TLC spot of a diameter of 1 mm and provides the These authors [194] provided also convincing evidence that the
possibility to resolve different components that could never be re- fluorescent dye ‘‘primuline” that is widely used in lipid research as
solved if the complete spot is scraped from the TLC plate. However, it binds non-covalently to the fatty acyl residues [179] may be used
there are three important problems that must be solved [22]: as a non-destructive, TLC/MALDI-compatible staining agent. Similar
data were independently obtained by another group that addition-
1. The matrix must be supplied in a very homogeneous fashion ally introduced ‘‘vibrational” cooling [195]. The term ‘‘vibrational
and in the form of very small droplets since otherwise the cooling” describes the desorption process in the pressure range,
achievable resolution would be compromised by the spreading where ‘‘cooling” of the excess energy of the generated ions is
of the matrix solution on the TLC plate as well as the formation achieved. Under these conditions, fragmentation of labile analytes
of large matrix–analyte crystals. can be minimized. This is normally performed by introducing an in-
2. The majority of commercially available MALDI-TOF devices are ert gas (e.g. N2) under a moderate pressure into the MS device.
currently equipped with UV lasers (k = 337 nm). UV irradiation In a recent study 2,5-dihydroxybenzoic acid (DHB) in acetoni-
does not penetrate very deeply into the silica layer and, thus, trile/water (1:1, v/v) was found very useful as matrix for the anal-
primarily compounds near to the surface of the TLC plate are ysis of glycosphingolipids because in this solvent mixture DHB is
detected. Due to these problems, quantitative aspects have highly soluble and this solvent mixture has a relatively high sur-
not yet been comprehensively addressed. This is a particular face tension leading to reduced analyte spreading. Sensitivities be-
problem regarding the fact that different compounds are tween 25 and 50 pmol could be obtained under these conditions. It
located at different silica gel depths in dependence on their should be noted that the application of highly concentrated matrix
RF-(retardation factor)-values. Therefore, IR-MALDI seems a solutions is very important because a larger excess of matrix over
suitable wayout because the IR radiation penetrates much dee- the analyte in comparison to standard MALDI (i.e. the use of a
per into the silica gel than UV. stainless steel target) is required for direct TLC/MALDI coupling
3. The achievable mass spectrometric resolution as well as mass in order to minimize fragmentation of the analyte [22].
accuracy is surely much lower if spectra are recorded directly A combination between MALDI, TLC and antibody detection was
from a TLC plate in comparison to a standard metal MALDI tar- also recently suggested [196,197]: these authors used the follow-
get. However, this is a minor problem in the field of lipid anal- ing workflow: (a) TLC separation of cancer-associated glyco-
ysis (in comparison to, e.g. tryptic digests of proteins) because sphingolipids (GSLs) from human hepatocellular and pancreatic
the smallest mass difference within a certain lipid class that tumors (b) their detection with oligosaccharide specific proteins
must be still resolved by MS is 2 amu, corresponding to one and (c) the direct in situ MS analysis of the GSLs previously de-
double bond. tected by the antibody. Detection limits of less than 1 ng of immu-
nostained GSLs could be obtained. It is a particular advantage of
5.1. Glyco- and sphingolipids this approach that crude lipid extracts of biological origin can be
directly used for TLC-IR-MALDI-MS and no laborious, previous
The majority of applications of TLC/MALDI have so far been ded- GSL purification is needed.
icated to the analysis of glycolipids, while phospholipids have been A ‘‘TLC-Blot-MALDI-Imaging” method was recently also sug-
investigated to a much lesser extent [22]. gested [198] to visualize individual molecular lipid species. This
In a rather early attempt [191], native glycosphingolipids were method was indicated to have higher sensitivity than common
separated on a conventional TLC plate and subsequently heat- staining methods and allows for direct visualization of all relevant
transferred to a different membrane consisting of polyvinylidene lipids within a linear range of approximately one order of magni-
difluoride (PVDF) where the MALDI measurements were per- tude. The important field of glycolipid analysis – particularly by
formed. This was done in order to remove impurities that are combined TLC/MALDI has been recently comprehensively re-
potentially coming from the TLC material and because spectral viewed [199].
quality and particularly the achievable sensitivity can be highly One important problem that is not only of relevance to TLC/
improved under these conditions. MALDI but as well to MS imaging of biological tissues stems from
In 2004, it could be shown that the analysis of gangliosides is the interference of matrix peaks with the signals of the molecules
possible by direct TLC/MALDI without major fragmentations of of interest. In order to overcome this problem, it was suggested to
the analyte [192]. In order to enhance the stability of the generated use graphite-assisted laser desorption/ionization (GALDI) MS and
ions, these authors used a relatively high gas pressure to allow col- this technique has already been successfully applied to the analysis
lisional ‘‘cooling” of the generated ions [192]. Recently is was also of cerebrosides in a total brain lipid extract of high complexity
shown that glycolipids from brain can be analyzed by using a very [200]. Beside the lack of typical ‘‘matrix” peaks it could also be
simple equipment, i.e. a commercially available MALDI-TOF MS de- shown that GALDI suffers far less from suppression effects, for in-
vice equipped with a standard nitrogen laser [193]. The majority of stance, the suppression of cerebrosides by other dominant lipid
TLC/MALDI studies of glycolipids have used UV lasers. However, species, particularly PC.
there are also some studies where infrared lasers were applied. In addition to positive ion detection, (negatively charged) sulfa-
These are primarily available on homebuilt MALDI devices but tides could be also easily detected using a thin layer of graphite.
commercially only on special request. Er:YAG (Erbium-doped yt- Thus, graphite does not only enable the recording of positive but
trium aluminum-Garret) IR lasers are normally used and have also of negative ion spectra although details of the basic ionization
the considerable advantage that IR radiation penetrates deeper have to be elucidated. So far, however, 9-AA seems to be the matrix
into the sample than UV radiation. Therefore, bringing the com- of choice to record negative ion MALDI mass spectra [201].
468 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

5.2. Glycerophospholipids ids separated on a TLC plate by MALDI-TOF MS was only recently
attempted by two different methods. One approach is based on
Much less information is so far available regarding the direct the use of an IR laser and glycerol as matrix [150]. This approach
MALDI/TLC MS analysis of phospholipids: the direct analysis of lip- has the significant advantage that IR radiation penetrates deeper

Fig. 16. Expanded region of a TLC-separated egg yolk extract and the corresponding positive ion MALDI-TOF mass spectra recorded directly from the indicated positions on
the plate. Only the relevant mass regions of each PL class are shown and assignments are provided directly in the individual traces. Data given in parentheses correspond to
theoretical masses and were introduced to enable comparison with the experimental data in selected cases. Please also note that the PE fraction provide different spectra,
depending on the position where the laser hits the PE spot. The only marked fragmentation is the loss of the head group of SM (leading to m/z = 677.5). Reprinted with
permission from Journal of Planar Chromatography 22 (2009) 35–42 [203].
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 469

into the TLC plate, but simultaneously confers the disadvantage fibrosis patients [205]. This is a clear indication that TLC/MALDI
that abundant glycerol adducts (and to a minor extent even NaCl data can be basically quantitatively analyzed although further at-
adducts) of the PLs of interest are detected and complicate inter- tempts are necessary to establish more useful and sensitive matrix
pretation of the spectra. Another problem is that IR lasers are com- compounds – particularly for negative ion detection directly on the
mercially barely available so far. Therefore, another approach used TLC plate [131,206].
a readily available N2 laser and standard DHB as matrix [202]. Due to the much higher experimental time that is required to
One selected lane of a TLC-separated hen egg yolk extract and record an MS image, 1D slices are normally investigated in the case
some selected MALDI mass spectra are shown in Fig. 16. of a developed TLC plate. However, 2D MS images can be also easily
Two facts are particularly remarkable in the context of Fig. 16. acquired and this is shown in Fig. 17.
First, even rather minor fractions of, e.g. phosphatidylinositol (PI) MALDI MS imaging, a fast developing application of MS will be
can be easily analyzed and give spectra with a convincing signal- discussed more comprehensively in the following section.
to-noise (S/N) ratio. As PI makes out only about 0.5% of the total
PLs of egg yolk [202], this clearly proves the significant dynamic
range of more than two orders of magnitude. Second, the lower 6. MALDI MS imaging
and the upper part of the PE spot provide significantly different
mass spectra, whereby PEs with longer fatty acyl (in particular Microscopic methods and radiological investigation techniques
arachidonoyl) residues are found in the upper and PEs with shorter (computer tomography, magnetic resonance imaging and sonogra-
acyl residues in the lower part of the spot. This is a clear indication phy) are well-known imaging methods. These methods have been
that even the separation quality obtained under normal phase con- established for several decades, whereas MS imaging is a rather
ditions is sufficient to allow the differentiation of fatty acyl resi- new, but very promising approach – not only in the basic sciences
dues if MS is used for subsequent analysis [203]. but as well in clinical diagnosis [207]. Although there are already
Regarding detection limits, both approaches [150,202] provided other MS imaging methods based on desorption electrospray
comparable results (about 400 pmol for PC detection) and, there- (DESI) MS [208] or secondary ion mass spectrometry (SIMS)
fore, both approaches might be useful for routine lipid analysis. [209] available (for a timely review see [210]), we will focus here
This particularly holds as surprisingly good mass accuracies (about exclusively to applications of MALDI MS. However it should be
100 ppm) and mass resolutions (about 3000) could be obtained noted that DESI MS has clear advantages if the analysis of smaller
that are absolutely sufficient for the differentiation of individual lipids is of interest. For instance, it could be convincingly demon-
PL species. strated upon the DESI image analysis of thin tissue sections of 68
Very recently, an automatic routine for the spatially-resolved samples of human prostate cancer and normal tissues that choles-
screening of a developed TLC plate became available and was al- terol sulfate (m/z = 465) is a useful marker of prostate cancer [211].
ready successfully applied for the investigation of the lipid compo- Although common (commercially available) UV lasers penetrate
sition of human mesenchymal stem cells [204] as well as plasma only a few lm into a solid sample such as thin tissue slices [20],
PL signatures associated with respiratory disease severity in cystic such (for instance histological) slices can be directly analyzed by
MALDI-TOF MS in order to obtain a two-dimensional concentration
profile of the molecules in or at the surface of this tissue. The data
source for creating such images is a set of mass spectra acquired
stepwise with a certain spatial resolution and the intensities of
the individual conventional mass spectra are afterwards trans-
formed into a greyscale – the MS image. Although the majority of
MS suppliers offers nowadays complete imaging solutions, some
special 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 the
quality of the image and only such metabolites can be displayed
that provide a reasonable conventional mass spectrum. This
means that molecules are the easier detected the higher their
ionization yields and the higher their concentration within
the tissue slice. This surely makes lipids more easily detectable
than high mass proteins.
2. MS imaging is quite an expensive method. Handling huge
amounts of data in a very short time (laser frequencies of the
order of kHz are nowadays rather common) requires excellent
computer hardware and data analysis software. Additionally,
lasers with a high lifetime are required: imagine a sample of
1 cm2. If this sample is scanned with a resolution of only
100 lm, 10,000 individual mass spectra have to be recorded.
If only 100 laser shots are averaged for each mass spectrum, this
means that one million laser shots are necessary for a single
image.
Fig. 17. Positive-ion MALDI-TOF mass spectrometric image (left) and densitometric 3. The achievable image quality is significantly dependent on the
image (right) of a developed HPTLC plate containing lipids from hens’ egg yolk. sample preparation and particularly on the even spotting of
Assignments of the individual spots and the m/z values of the signals that were used the tissue slice with the matrix. Already small inhomogeneities
for image analysis are provided. Please note that two different spots may be
observed for most lipid classes and that lipids with ‘longer’ fatty acyl residues can
of the matrix deposition will seriously compromise the image
be easily differentiated form those with shorter fatty acyl residues. Reprinted with quality. Of course, this also holds for slight fluctuations of the
permission from Journal of Planar Chromatography 22 (2009) 35–42 [203]. laser fluence.
470 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475

Many of the points that have to be carefully considered in the [213]. In addition to brain (that is often investigated because this
context of successful MALDI MS imaging were recently summa- tissue is particularly rich in a large variety of lipids [193]) the lipid
rized by Bernhard Spengler, one of the pioneers in this field [212] compositions of many different tissues have already been success-
in [20]. Therefore, we do not want to give so many methodological fully investigated by MALDI-TOF MS imaging [217] and these
details but will focus on some selected applications. compositional data compared with results from established bio-
The simplest approach to perform MALDI MS imaging is to fix chemical methods. A surprisingly good agreement between both
the tissue slice onto the MALDI target and to cover it with matrix. methods is often found.
The homogeneous spotting of the matrix is unequivocally the most Therefore, tissue samples can be analyzed for their lipid compo-
critical point and many efforts for its improvements were and still sitions by direct MALDI-TOF MS without sample workup [218].
are undertaken [213] and devices for homogeneous matrix deposi- It seems that the application of liquid ionic matrices provides
tion onto the sample of interest by sophisticated spraying tech- superior image qualities in comparison to conventional crystalline
niques are nowadays commercially available. matrices due to the more even distribution of the matrix on the tis-
Although not originally in the focus of interest, different classes sue surface [219]. This is the most important prerequisite for using
of lipids are primarily detected if native tissue slices are investi- MALDI-TOF MS as a (quantitative) imaging technique [220], i.e. to
gated by MS imaging because these species ionize particularly well obtain spatially-resolved information about the PL distribution
and lipids are abundant constituents of all tissues. Thus, a much within a given sample. Unfortunately, the achievable resolution
higher interest in lipid analysis by MALDI-TOF MS has been obvi- is normally not sufficient to obtain images with cellular resolutions
ous over the last five years [214]. This particularly holds because [20]. Thus, the combination between MALDI and SIMS imaging is a
there is increasing evidence that not only proteins but also certain very promising approach: while SIMS is capable of providing
lipid classes are useful as disease markers. For instance, it has re- images with a subcellular resolution, MALDI is unequivocally the
cently been suggested that LPC may be used as a marker molecule method of choice to detect lipids with relatively high masses such
of ovarian cancer [215] and that differently saturated LPCs possess as cardiolipins [221].
either inflammatory or anti-inflammatory properties [216]. A typ- Although the lipid compositions of many different tissues (e.g.
ical example of a positive ion MALDI image of a mouse brain is brain [218], eye lenses [220], and dystrophic muscle [222]) have al-
shown in Fig. 18 ready been examined by MALDI-TOF MS, the most intriguing suc-
Fig. 18 shows the type of high quality images that can be gen- cess of MALDI imaging was to prove that it is possible to
erated for individual lipid species in tissues using MALDI MS, in differentiate tumor tissue from the ischemic and necrotic areas
which clear anatomical distribution of these species are observed. of the lesion [223]. Already this selected example clearly indicates
This figure compares an optical image of a mouse brain sagittal that MALDI imaging will have great future developments because
section with three 2D ion intensities maps generated by MALDI it turns increasingly out to be useful for clinical diagnostics. A com-
imaging. The MALDI images were recorded in positive-ion mode prehensive review dealing with MS imaging - not only of the re-
and sublimation of the matrix was employed to coat the tissue lated lipid aspects - is available in [122].

Fig. 18. (a) Mouse brain, sagittal section, stained with Oil Red O. Section was taken 100 lm lateral from the imaged slice. A tear in the tissue occurred in the pons region
during the tissue cutting process. (b) Mass spectrometric image of mouse brain sagittal section acquired in positive-ion mode, displayed at m/z = 760.6. The image shown was
acquired with 50 lm plate movements, and is displayed smoothed, relative to the intensity scale shown at the left of the image. The white bar indicates a 1 mm distance. (c)
Mass spectrometric image of mouse brain sagittal section acquired in positive-ion mode, displayed at m/z = 826.6. The image shown was acquired with 50 lm plate
movements and is displayed smoothed with the scale intensity normalized to the most abundant ion at m/z = 760.6. (d) Mass spectrometric image of mouse brain sagittal
section acquired in positive-ion mode, displayed at m/z = 834.6. The image shown was acquired with 50 lm plate movements and is displayed smoothed with the scale
intensity normalized to the most abundant ion at m/z = 760.6. Reprinted with permission from J. Am. Soc. Mass Spectrom. 18 (2007) 1646–1652.
B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475 471

7. Summary ful advice of Dr. Suckau and Dr. Schürenberg (Bruker Daltonics,
Bremen) as well as Dr. Thorsten Jaskolla (Goethe University, Frank-
Unequivocally there is considerable interest in lipid analysis furt) is gratefully acknowledged.
and it is expected that this interest will increase in the future be- This work was supported by the German Research Council (DFG
cause many diseases are recognized to be accompanied by altera- Schi 476/12-1 and FU 771/1-1 as well as TR 67 project A2) and the
tions in the lipid compositions of either body fluids or tissues or Federal Ministry of Education and Research of the Federal Republic
even both. Thus, lipids are of unequivocal diagnostic relevance. of Germany (‘‘The Virtual Liver”, 0315735).
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