Progress in Lipid Research 49 (2010) 450–475

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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
We are currently living in an ‘‘omics” time period [1]. Although
this list is surely not complete, there were already proteomics,
genomics, lipidomics, metabonomics, metagradomics, metallomics
and – rather recently – interactomics. As these approaches provide
an immense amount of data, bioinformatics is an indispensable
counterpart leading to ‘‘systems biology” initiatives [2] that may
help to get further insights into complex metabolic networks of
biological systems.
As this review is dedicated to lipids and their analysis, a short
definition of ‘‘lipidomics” is necessary: according to a recently pro-
vided definition [3], ‘‘lipidomics” can be defined as ‘‘the full charac-
terization of lipid molecular species and of their biological roles
with respect to expression of proteins involved in lipid metabolism
and function, including gene regulation”. The combination of all
these complex aspects is clearly a very challenging task, but the
first step is obviously the qualitative and quantitative knowledge
of the lipid composition of an unknown sample.
Many ‘‘omics” applications are based on the application of mass
spectrometry (MS) and would not have been possible without the
significant progress achieved in this field during the last decades.
MS is an incredible powerful method and may be regarded as an
indispensable tool in physics, chemistry, biochemistry and
(increasingly) medicine and particularly clinical diagnosis [4].
The history of MS began more than one century ago and was ini-
tially related to the discovery of the different isotopes of the chem-
ical elements [5] and, thus, primarily a task of physicists. For
instance, Thompson was awarded the Nobel Prize (of Physics) in
1906 for his work on ‘‘Conduction of Electricity through Gases”.
In contrast, the history of the success of MS in biology and life
sciences is much shorter and directly related to the discovery of
new and gentle ionization methods: over many decades electron
ionization (EI) was exclusively available as ionization method [4].
EI makes use of fast electrons to ionize the analyte molecules in
the gas phase by the removal of one electron. Although this tech-
nique was (and still is) unequivocally suitable for the investigation
of small and/or volatile compounds, EI normally fails if larger mol-
ecules with low volatilities are to be analyzed: although it could be
shown that the molecular ion (M ? M+ + e) of phospholipids is
basically detectable if EI is used for ionization of the sample, the
obtained results were not very convincing [6] and fragment ions
are much more abundant in comparison to M+ making this tech-
nique less suitable for the analysis of mixtures. Therefore, the
prime application of EI MS in the context of lipids is nowadays nor-
mally the analysis of free fatty acids that can be obtained by sapon-
ification of lipids. This also holds for oxidation products derived
from free fatty acids and their metabolic products such as throm-
boxanes or leukotrienes [7]. GC/MS is even nowadays a highly
established and widely used method for the quantitative analysis
of relatively apolar compounds. Unfortunately, sample preparation
is quite cumbersome and time-consuming. Because of these disad-
vantages, the invention of soft-ionization methods, enabling the
analysis of molecules refractive to EI, must be regarded as a real
milestone in the history of modern mass spectrometry [8].
1.1. Soft ionization mass spectrometric methods
The differentiation between ‘‘hard” and ‘‘soft” ionization MS
methods is a matter of philosophy and depends to a significant ex-
tent on the analyte of interest and its properties. However, all mod-
ern ionization methods may be regarded as ‘‘softer” than EI [9].
Methods enabling the ionization of compounds refractive to elec-
tron collision ionization comprise gas phase ionization techniques
such as chemical ionization (CI) that was discovered in 1913 by
Thompson [10], field desorption (FD) and field ionization (FI),
methods based on particle bombardment such as fast atom bom-
bardment (FAB) or secondary ion MS (SIMS) [11]. Each of these ion-
ization methods has its individual strengths and weaknesses, but
the application of these methods is normally limited to special
problems or selected analytes [4]. Only chemical ionization (often
in combination with atmospheric pressure, APCI) is used to a high-
er extent for the analysis of lipids. For a recent review of applica-
tions of CI in lipid research see [12], while a survey of the
advantages and drawbacks of the different ionization techniques
is available in [13].
Today, electrospray ionization (ESI) and matrix-assisted laser
desorption and ionization (MALDI) MS are most often used and
the majority of commercially available MS devices is equipped
with one (or maybe even both) of these ion sources. As a maximum
of information is obviously achievable if both techniques are com-
bined, they should be regarded as complementary but not as com-
petitive. The importance of these both ionization methods was
obviously also the reason why the inventors of ESI and MALDI,
i.e. Fenn and coworkers [14] and Tanaka and coworkers [15],
respectively, shared the Nobel Prize for Chemistry in 2002 [16].
452 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
We will not provide here a comprehensive survey of the funda-
mentals of ESI MS (for reviews dedicated to ESI MS analysis of lip-
ids see [17,18]), but will focus on applications of MALDI MS and the
related methodology. A review with a similar topic was already
published by our group in 2004 in ‘‘Progress in Lipid Research”
[19]. However, many important improvements regarding potential
applications, matrix optimization, quantitative data analysis and
more detailed characterization (particularly by MS/MS) of the
different lipid classes have been achieved since that time and an
update of our previous review seemed necessary. In our opinion
the most important developments can be summarized as follows:
1. Although details of ion generation are still not yet completely
understood, some progress in understanding this important
aspect has been achieved. This is very important because fur-
ther theoretical insights will help to improve the performance
of the MALDI method. A comprehensive survey of theoretical
aspects is available in [20].
2. Some new and improved matrix compounds were introduced
that provide either higher sensitivities or higher reproducibili-
ties than previously used matrices [21]. We will emphasize here
that some lipid-derived compounds are exclusively detectable if
the most appropriate matrix is used.
3. The coupling between MALDI MS and chromatographic separa-
tion techniques is unequivocally a ‘‘hot” topic of current
research. This particularly concerns the combination between
MALDI MS and thin-layer chromatography (TLC) that helps to
overcome many problems related to mixture analysis in a sim-
ple but elegant way [22].
4. MS imaging is nowadays an established method to monitor the
distribution of certain molecules within a tissue. As lipids ionize
particularly well, they are very easily detectable under these
conditions and this fact has pushed the interest in lipid analysis
by MALDI MS considerably [23].
The continuously increasing interest in MALDI MS as well as its
applications in lipid analysis is clearly reflected by the data given in
Fig. 1.
It must be explicitly stated that we do not want to suggest MAL-
DI MS as the best method of lipid analysis. However, during the last
centuries there were a lot of ‘‘omics” initiatives and many MALDI
devices were purchased – particularly for protein and peptide
analysis. It is our aim to convince the reader that these instruments
are not exclusively useful in the proteomics field, but the same
instruments can be readily used for the analysis of lipids. There
is surely no need to buy an additional device but the available
MALDI devices can be used without the need of major alterations!
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.
1.2. Fundamentals of MALDI mass spectrometry
A detailed survey of methodological aspects of MALDI MS is
available in the excellent book by Franz Hillenkamp and Jasna
Peter-Katalinić [20]. Therefore, theoretical fundamentals of MALDI
MS will be only shortly discussed in this review. However, a short
introduction is necessary for the less experienced reader.
MALDI MS is based on the utilization of a ‘‘matrix” that initially
absorbs the energy of the laser and mediates the generation of ions.
Although inorganic compounds such as graphite [24] or metal
oxide particles [25] may be also applied as matrix, small organic
molecules are used in the majority of cases and will be, thus, nearly
exclusively discussed here.
Of course, the suitability of a certain compound as matrix is
determined by the type of the laser and its emission wavelength.
Although many different lasers, including Nd:YAG (k = 355 or
266 nm) [26], excimer [27] or CO2 lasers [28] were already success-
fully applied, in the lipid field N2 lasers are primarily used.
Although IR lasers provide some advantages (see below), we will
focus here nearly exclusively on UV lasers with an emission wave-
length of 337 nm because the majority of commercially available
MALDI devices are equipped with this laser type. This is the reason
why the majority of the so far available lipid data were obtained
with UV lasers. A more comprehensive discussion of the role of
the matrix is available in [29].
When the pulsed laser beam hits the sample (normally co-
crystals of the matrix and the analyte), its energy is primarily ab-
sorbed by the matrix that is present in a vast excess over the analyte
(a 100–100,000-fold excess of the matrix is typically used). Conse-
quently, the matrix is vaporized, carrying intact analyte molecules
into the vapor phase. A simplified schema of the processes occurring
in a typical MALDI-TOF mass spectrometer is shown in Fig. 2 [19].
During the expanding process of this gas cloud, ions (e.g. H+ and
Na+) are exchanged between the matrix and the analyte, leading to
the formation of charged analyte molecules. These analyte ions are
called ‘‘adducts” or ‘‘quasimolecular ions” (sometimes the term
‘‘pseudomolecular” ions may be also found although the use of this
term is discouraged by the IUPAC). Beside cation generation, an-
ions can also be generated by abstracting H+ or Na+ from the ana-
lyte. The ratio between the cation and the anion yield is
determined by the (gas phase) acidities of the analyte and the ma-
trix [20] and fundamentals of the ion formation process were re-
cently comprehensively reviewed by Zenobi and Kochenmuss
[30,31]. Recording positive-ion mode spectra is much more com-
mon and it even seems (although not yet investigated in detail)
that many MALDI mass spectrometers detect negative ions less
sensitively than positive ions.
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
The most important difference between ‘‘molecular (radical)
ions” (generated in conventional EI mass spectra) and ‘‘quasimo-
lecular ions” is the observed mass. As molecular ions are generated
by the abstraction of one electron (the mass of which can be ne-
glected for the majority of applications because it accounts for just
about two-tenths of a percent of the mass of a proton) from the
analyte, the mass of the generated ion (or the observed m/z ratio)
corresponds to the mass of the analyte. In contrast, quasimolecular
cations are generated by the addition of a cation to the analyte.
Therefore, the mass of the quasimolecular ion is characteristically
higher (normally 1 amu or 23 amu corresponding to the mass
of H+ or Na+, respectively) in comparison to the analyte molecule
[32].
Despite its profound importance, the process of ion generation
is so far only poorly understood and many papers are currently
dealing with this important topic. For instance, one recent issue
of European Journal of Mass Spectrometry (2006, Volume 12, Issue
6) was exclusively dedicated to the topic ‘‘Mechanisms of MALDI”.
Despite this obvious lack of knowledge, it is sure that singly-
charged ions are primarily generated [33] and, therefore, the actual
measured quantity, the mass-to-charge ratio (m/z) may be re-
placed directly by the monoisotopic mass of the analyte molecule –
plus or minus the mass of the ion required to generate a charge.
The use of ‘‘Thompson” [Th] as unit of the mass over charge ratio
was suggested nearly 20 years ago [34] but is not yet widely estab-
lished. A detailed discussion of this aspect is available in [35].
After being formed, ions are accelerated in an electric field (typ-
ically of the order of 20 kV). After passing a charged grid, the ions are
drifting freely over a field-free space where mass separation is
achieved: low mass ions arrive at the detector in a shorter time than
high mass ions [13]. This is the most simple, ‘‘linear” geometry of a
TOF analyzer that is normally used for the analysis of larger mole-
cules because in this case high resolution is less important than high
sensitivity. Resolution and peak widths may be improved by using a
reflectron [20]. The reflectron enlarges the flight path and helps to
compensate differences in the initial velocities of the ions during
the ablation process. All spectra shown in this review were recorded
on a MALDI-TOF device equipped with a reflectron. Using commer-
cial MALDI-TOF devices with reflectron configuration, mass resolu-
tions of about 10,000 and mass accuracies higher than about 30 ppm
can be routinely achieved. This is rather poor in comparison to the
best available mass spectrometers nowadays which provide mass
accuracies lower than 1 ppm and also much higher resolutions. Such
high quality mass spectra are desirable in the context of proteomics,
where an increased mass accuracy helps to unequivocally assign un-
known (tryptic) peptides because the number of hits upon database
searching is significantly reduced [36] if the mass can be provided
with higher accuracy.
Matrix Nitrogen Laser
Sample (337 nm) Charged
Plate
Sample
Target
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].
453
In the context of lipids, however, very high mass accuracies do
not really provide a much larger extent of information because the
smallest mass difference within one given lipid class is 2 amu, i.e.
one double bond. We will show below that there are some simple
chemical/biochemical methods (addition of certain salts or enzy-
matic digest) to overcome problems related to potential signal
overlap and a MALDI device with a high resolving power is not
absolutely needed for routine lipid analysis.
It should be noted that there is no absolute need to combine a
MALDI ion source with a ‘‘TOF” mass analyzer. However, as MALDI
is used in the majority of cases for the detection of rather large
molecules, the TOF detector is very popular because it has a nearly
unlimited mass range [20]. An additional reason is the pulsed (not
continuous) ion generation of MALDI that is most suitable for the
TOF mass analyzer. A more comprehensive survey of typical mass
analyzers as well as their individual advantages and drawbacks is
available in [13,20].
2. The role of the matrix – some practical considerations
Although there were already different attempts to predict the
suitability of an unknown chemical compound as MALDI (UV) ma-
trix by theoretical considerations, most matrices were (and actu-
ally are) found by accident or by a ‘‘try and error” approach. A
more detailed discussion of these aspects is beyond the scope of
this review and we will focus exclusively on some selected practi-
cal aspects. A ‘‘good” MALDI UV matrix in the practical sense is
characterized by the following properties:
1. A suitable matrix should provide an excellent signal-to-noise (S/
N) ratio for the peaks of the analyte of interest, i.e. a high sen-
sitivity should be achievable.
2. The matrix should provide a high absorbance at the emission
wavelength of the laser. Thus, the required laser fluence should
be as low as possible because enhanced laser fluence leads to
enhanced analyte fragmentation and/or poorly resolved mass
spectra.
3. An optimum matrix is characterized by low background (i.e. the
signals of the matrix should be very small) in order to avoid
interferences between the matrix and the analyte ions. It is
important to note that oligomers of the matrix are often gener-
ated in the gas phase. Therefore, most matrices provide signals
at m/z ratios much higher than their actual molecular weight!
4. Finally, an ideal matrix should provide only a single adduct of
the analyte and exhibit a weak tendency to cluster formation
because analyte–matrix clusters may seriously complicate data
analysis [37].
Reflector
Detector
mass to charge
(m/z)
Reflector
("electrostatic Mirror") Linear
Grid Detector
Heavy IonsLi ght Ions
mass to charge
(m/z)
Field-free time-of-flight
454 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
As the vast majority of commercially available MALDI devices
are equipped with nitrogen lasers, the focus of this review will
be on matrices compatible with N2 lasers (337 nm), whereas infra-
red lasers and the corresponding matrices (such as glycerol or
succinic acid) will be treated only briefly. There are basically five
typical requirements that must be met by a powerful MALDI
matrix and some selected UV MALDI matrix compounds are shown
in Fig. 3.
(a) The matrix must exhibit a strong absorption at the laser
emission wavelength, i.e. in the case of a UV laser normally
at 337 nm. This is the reason why nearly all common organic
matrices contain an aromatic ring system with delocalized p
electrons. Inorganic matrices such as graphite or metal salts
will only be shortly discussed here because they are scarcely
applied to the field of lipid analysis so far [38]. As expected,
the ionization efficiency increases if the absorption coeffi-
cient of the matrix at the laser wavelength increases and this
is one reason why among the different isomers of dihy-
droxybenzoic acids (DHB), only the 2,5 isomer that provides
the most marked absorption at 337 nm (but not the other
isomers) is commonly used as MALDI matrix [39]. Although
the solution UV absorption of the matrix is determined in
many studies, it must be emphasized that exclusively the
UV absorption in the solid state determines the absorption
properties under MALDI conditions. Absorptions determined
in the liquid and in the solid state may be significantly dif-
ferent [39].
(b) The energy absorption and the subsequent evaporation of
the matrix must lead to the generation of ions from the ana-
lyte of interest. This is often believed to be the reason for the
use of carboxylic acids (many matrices are derived from cin-
namic or benzoic acids) as matrix compounds because
Fig. 3. Survey of some important UV MALDI matrices that are often used in the field
of lipid analysis. The molecule classes from which the individual matrices are
derived are also indicated. Please note that this is only a selection of the (in these
authors opinion) most important matrices in the field of lipid analysis.
organic acids are acidic and, thus, capable of triggering the
generation of H+ adducts. As sodium is an ubiquitous ele-
ment, however, there are normally also Na+ adducts in addi-
tion to H+ adducts. In addition to the enhanced generation of
H+ adducts, there is another practical reason to use carbox-
ylic acids: due to the presence of the aromatic ring system,
typical MALDI matrices are well soluble in organic solvents.
As MALDI is particularly used for the analysis of polar mole-
cules that are only scarcely soluble in organic solvents, water
is one important constituent of the most common solvent
systems: the presence of polar groups (such as carboxylic
acids) increases the solubility of the matrix in polar solvents
[13].
(c) The matrix should be stable under high vacuum conditions.
Although this sounds trivial, there are many potentially use-
ful MALDI matrices that do not – or not sufficiently – fulfill
this criterion. As most MALDI devices make use of high vac-
uum conditions (normally about 1  109 bar) many com-
pounds tend to sublime. The loss of the matrix under high
vacuum conditions may be one important reason why
MALDI mass spectra show time-dependent changes [40].
(d) The matrix should isolate the generated ions and prevent the
generation of analyte clusters, for instance, dimer formation.
This is the prime reason why a significant excess of the
matrix over the analyte is normally required in order to
obtain optimum results. Additionally, the laser fluence
should be kept as high as needed but as low as possible
because matrix analyte clusters are increasingly generated
at elevated laser fluences [19].
(e) The matrix and the analyte should give homogenous co-
crystals. Improvement of homogeneity is clearly science of
its own and depends – beside the analyte, the matrix and
the solvent system – on the method of sample preparation.
This important topic would be a review of its own and has
been reviewed elsewhere [41] but with the focus on polar
molecules. Briefly, the simpler the sample preparation
method, the lower is the homogeneity of the matrix/analyte
mixture. The most frequently used ‘‘dried droplet” method,
i.e. the successive deposition of matrix and analyte are 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
important matrices in the lipid field will be discussed in more de-
tail in the subsequent paragraphs of this review. A more complete
survey is available in [29].
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
However, we would like to emphasize that lipids offer a consid-
erable advantage in comparison to polar molecules: as already
indicated above, the majority of MALDI devices make use of a UV
laser emitting at 337 nm. This normally requires matrix com-
pounds with aromatic residues. Such compounds are only barely
soluble in water, but well soluble in organic solvents. Thus, spectra
of apolar molecules can be recorded in a single organic phase with-
out the need of adding water. This results in enhanced reproduc-
ibility and only relatively moderate shot-to-shot deviations [43]
that are often serious problems regarding the MALDI MS analysis
of polar molecules.
2.2. Inorganic MALDI matrices
As the matrix is normally applied in vast excess over the ana-
lyte, typical matrix peaks (often representing cluster ions with
higher masses than the original matrix compound) are normally
seen in the MALDI spectra – particularly in the smaller m/z range.
This may lead to reduced sensitivities and the potential suppres-
sion of analyte signals. Therefore, the application of inorganic com-
pounds that do not give any signals is a straightforward approach.
The very first application of an inorganic matrix was published
by Tanaka and coworkers [15] who used glycerol suspensions of
cobalt nanoparticles for the ionization of large molecules. The per-
formance of lm size metal and metal oxides such as Al, Mn, Mo, Si,
Sn, SnO2, TiO2, W, WO3, Zn and ZnO for the detection of small or-
ganic molecules was also tested by Kinumi [25] using polyethylene
glycol 200 and methyl stearate as selected ‘‘small” analytes. Higher
sensitivities in comparison to classical organic matrices could be
obtained and no interfering matrix background signals were de-
tected. Generally, decreasing particle size results in increased sen-
sitivity and, thus, nanoparticles instead of microparticles are
nowadays preferentially used. Although these metal or metal oxide
particles do not give any ‘‘matrix” peak, they have a very high melt-
ing point and, thus, enhanced laser fluences have to be used to
transfer them into the gas phase. This often results in enhanced
analyte fragmentation [44]. Another problem that hinders their
wider application is their limited commercial availability.
Carbon nanotubes could be also successfully used and it has
been shown that detection limits of certain analytes can be im-
proved for one or two orders of magnitude [45]. An additional
advantage of these materials is that they can be used to enrich cer-
tain apolar analytes due to their considerable hydrophobicity. Inor-
ganic materials such as silicon [46], graphite [47] and TiO2 [48]
have been used to coat the surface of the MALDI plate, and no addi-
tional matrices were used. Fabrication of complete MALDI plates
from porous silicon and graphite for matrix–less analyte detection
has also been reported [49]. Such inorganic oxides are especially
useful because their isoelectric points vary over a wide range from
acidic to basic. Therefore, the creation of positive or negative ions is
favored depending on the oxide choice.
Some selected applications of these matrices will be described
at the appropriate places in this review. However, inorganic 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
[52]. In this review we will deal exclusively with apolar com-
pounds and particularly with lipids and phospholipids that can
455
be found in living organisms. As we are sure that the readers of
‘‘Progress in Lipid Research” will know the related chemical struc-
tures, we will pay only minor attention to this point. Our previous
review in this journal [19] contained a survey of the related chem-
ical aspects and may be consulted if needed. Additionally, there
was a recent paper that gave a comprehensive survey of the classi-
fications of lipids [53].
3.1. Hydrocarbons and wax esters
As already indicated above, positive ion MALDI MS is much
more common than negative ion MALDI MS. In order to convert a
(neutral) molecule into a positively-charged ion, a cation, for in-
stance a proton must be added. Expectedly, this normally takes
place at sites with a high electron density, i.e. charged groups such
as phosphate or atoms with an electron lone pair such as nitrogen
or oxygen.
Nevertheless, even completely apolar compounds (without any
oxygen, nitrogen or sulfur heteroatoms) such as the unsaturated
hydrocarbon squalene (the structure of which is shown in Fig. 4),
a hydrocarbon containing several isoprene units, as well as its olig-
omerization products can be easily analyzed by MALDI-TOF MS
[54]. Squalene occurs in olive oil (about 0.8–12 g/kg) and has been
assumed to be related to the health benefits of vegetable oils and
is, thus, of significant nutritional interest.
Due to the apolarity of squalene, an auxiliary reagent such as
silver trifluoroacetate (AgTFA) is often added to the matrix, for in-
stance, DHB [55] in order to improve the yield of ions.
As many hydrocarbons contain olefinic residues and give, thus,
rise to a significant UV absorption, simple laser desorption (LD) MS
can also be used because an additional matrix is not absolutely
necessary and does not significantly improve the spectral quality.
This provides the significant advantage that there is no interfer-
ence with matrix peaks at all. However, direct LD analysis is impos-
sible if completely saturated hydrocarbons are to be analyzed as
these compounds lack sufficient UV absorptions.
Asphaltene fractions of crude petroleum [56] can be investi-
gated in a similar way and also in this case the MALDI and the
LDI spectra are of comparable quality. Additionally, applications
of MALDI-TOF MS to polyaromatic compounds [57] have also been
described although the insolubilities of these molecules conferred
some problems. These problems were solved by using a new sam-
ple preparation method consisting of mechanically mixing analyte
and matrix without the necessity to use solvents [58]. Finally,
7,7,8,8-tetracyanoquinomethane (TCNQ) was shown to possess
superior properties in comparison to other matrices [59]. A more
comprehensive survey about LD MS is available in [60] and this re-
views covers also aspects of desorption/ionization on silicon (DIOS)
as well as carbon-based microstructures. Finally, a quite old, but
nevertheless excellent review dealing with LD MS is available in
[61].
Fig. 4. Chemical structure of the physiologically important hydrocarbon ‘‘squa-
lene”. Virgin olive oil is a rich source of this hydrocarbon and the squalene content
of vegetable oils is often assumed to be important for the related health benefits.
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
matrix for hydrocarbon analysis than the free acid DHB: LiDHB
provides less pronounced fragmentation and higher sensitivity
[62]. Similar data were also obtained in the case of wax esters
[63] and insect cuticular hydrocarbons [64], whereby saturated
and unsaturated hydrocarbons with more than 70 carbon atoms
could be detected. In a very new paper it was shown that the dis-
tribution of different wax esters and other apolar compounds can
also be investigated by MALDI imaging MS [65]. Of course, there
are also many different apolar polymers the analysis of which
might be of significant interest. These compounds will not be dis-
cussed here but the interested reader is referred to the excellent
review by Batoy and coworkers [66].
3.2. Plant pigments
Since a pioneering MALDI work in 1996 [67], only very moder-
ate efforts were undertaken to study chlorophylls and other plant
pigments [68]. This is somewhat surprising because these pig-
ments play a very central role in the plant metabolism, particularly
regarding photosynthesis. Using standard DHB matrix, such mole-
cules are easily detectable. However, they are only detectable sub-
sequent to removal of the central metal ion [69]. Chlorophyll a, for
instance, is detected at m/z = 871.5, although its monoisotopic
mass is 892.5 (for chemical structure see Fig. 5).
The observed mass difference may be explained by the addition
of three H+ and the loss of Mg2+ leaving a single positively-charged
ion. A very recent study [70] provided evidence that the loss of the
central ion can be avoided if terthiophene is used as the MALDI
matrix. In contrast, however, fragmentation of the phytol-ester
linkage was more pronounced in the presence of the terthiophene
matrix. It seems likely that an enhanced laser fluence was needed
under these conditions due to the weaker UV absorption of this
matrix resulting in enhanced fragmentation.
3.3. Flavonoids and carotenoids
These compounds can be easily characterized by MALDI-TOF
MS although their tendency to give fragment ions seems strongly
dependent on the applied matrix. According to current knowledge,
Fig. 5. Chemical structure of chlorophyll, a very important plant constituent that
enables light fixation during the photosynthesis process.
only a minor extent of fragmentation is detectable if 20 ,40 ,60 -trihy-
droxyacetophenone (THA) is used as matrix, whereas DHB gives
much higher yields of fragment ions and is, thus, the matrix of
choice to record post source decay (PSD) mass spectra [71]. It
was also reported in the context of red wine analysis that quanti-
tative data can be directly achieved from the MALDI mass spectra
[72]. Due to the absorbance of these compounds in the UV range,
recording simple laser desorption spectra is a potential alternative.
It has been recently shown that direct imaging of plants is also pos-
sible [73]: as no matrix addition is required (that determines the
achievable resolution due to the size of the crystals), highly re-
solved (resolution about 10 lm) MS images can be obtained and
it could be shown that the highly specific distribution of important
flavonoids such as kaempferol, quercetin and isorhamnetin can be
imaged at the cellular level. Similar data can be also obtained if col-
loidal graphite is used as matrix [74].
In a very recent work it could be shown that flavonoids such as
quercetin or rutin may be used themselves as matrices for inor-
ganic metal complexes [75] and provide better results in compar-
ison to common crystalline matrices such as DHB.
3.4. Free fatty acids
The MALDI-TOF MS analysis of free fatty acids is quite difficult if
standard MALDI matrices are used. The most serious problem is the
overlap of the signals of the free fatty acids with matrix signals that
are particularly abundant in the low mass range. Of course, this is a
serious problem if fatty acids at low concentrations have to be ana-
lyzed. According to our best knowledge standard matrices such as
DHB or CHCA (a-cyano-4-hydroxycinnamic acid) are not suitable
for this purpose, but some methods as to how this problem can
be overcome have been suggested:
1. If meso-tetrakis(pentafluorophenyl)porphyrin (MTPFPP), the
structure of which is shown in Fig. 6 [76], is used as matrix,
the problem of signal and matrix overlap can be overcome.
MTPFPP has a relatively high mass and does not give signals
below m/z ffi 500 in the positive-ion mode. Thus, fatty acids
with typical masses between about 200 and 350 Da can be
unequivocally detected. This was demonstrated, for instance,
with fatty acid mixtures obtained from different vegetable oils
subsequent to alkaline hydrolysis [77]. As an excess of sodium
acetate was added, exclusively the Na+ adducts of the Na+ salts
of the released fatty acids were detected. Therefore, there are no
problems with the overlap of different adducts, on the one
hand, and differences in fatty acyl compositions (the H+ adduct
of arachidonic acid (20:4) results in the same m/z ratio as the
Na+ adduct of oleic acid (18:1)), on the other hand, could be
completely avoided. This method was also successfully used
for the analysis of complex fatty acid mixtures from different
biological samples, for instance, rat plasma [78]. However,
MTPFPP is so far not widely used because this matrix gives rise
to unknown artifacts. Typical examples of positive ion mass
spectra of selected free (saturated (6a) as well as unsaturated
(6b)) fatty acids recorded in the presence of MTPFPP are shown
in Fig. 6. Although all saturated fatty acids are detected with the
expected m/z ratios, a mass shift of 14 amu to higher masses is
observed if unsaturated fatty acids are investigated [79]. For
instance, oleic acid is observed at m/z = 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.
2. Fatty acids are acidic compounds and should be, thus,
easily detectable as negative ions – at least if a sufficiently
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
alkaline matrix is used. 9-Aminoacridine (9-AA) seems a
particularly promising matrix in this field due to its consider-
able basicity (the pKa is about 9.99) [80]. Another advantage
of 9-AA is the very moderate background [81] provided by
this matrix. It could be shown that the achievable sensitivity
is in the femtomolar range and linear detector response
could be obtained over two orders of magnitude. Therefore,
9-AA seems the matrix of choice for quantitative metabolomics
studies of negatively charged compounds – not only fatty acids
[82].
3. Very recently [83], it could be shown that 1,8-bis(dimethyl-
amino)naphthalene (DMAN), a ‘‘superbasic” compound with a
pKa of 12.21 [84] is a powerful matrix because it enables the
detection of fatty acids (saturated and unsaturated ones) in
low picomole amounts. Therefore, both, 9-AA and DMAN seem
more suitable than MTPFPP because all fatty acids can be easily
and accurately detected, while the MTPFPP porphyrin matrix is
obviously less suitable for unsaturated fatty acids (cf. Fig. 6b)
due to the +14 amu artifact and the lower achievable sensitiv-
ity. However, there are rumors that the use of DMAN may
decrease the sensitivity in the positive-ion mode significantly:
residual amounts of DMAN may bind all available protons and
reduce the achievable sensitivities by reducing the available
amount of this cationizing species.
4. Free fatty acids are also detectable if inorganic matrices such as
graphite [85] or porous silicon [86] (desorption/ionization on
porous silicon (DIOS MS)) are used. Although the achievable
sensitivity is rather poor, deprotonated fatty acids could be eas-
ily detected as negative ions. A more detailed survey of this
topic is available in [87].
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].
457
3.5. Cholesterol and cholesteryl esters
Although cholesterol is present in virtually all mammalian cells
and body fluids [43] as well as in combination with significant
amounts of cholesteryl esters (CE) and triacylglycerols (TAG) in
the lipoproteins of blood, only little interest has been paid to the
MALDI MS characterization of these molecules. One potential rea-
son is the commercial availability of enzymatic test kits that help
to determine both, cholesterol and cholesteryl ester concentrations
making MS methodology less important for the determination of
these molecules. Using established MALDI matrices such as DHB,
cholesterol is not detectable as the expected H+ adduct but only
subsequent to water elimination at m/z = 369.3 (M+H+H2O)
[88]. Although it has been shown that the cholesterol concentra-
tion in extracts of, e.g. human lipoproteins can be accurately deter-
mined by MALDI-TOF MS [89], the relatively small mass of
cholesterol confers problems: in the same manner as in the case
of free fatty acids, there is a considerable overlap between the cho-
lesterol peak and the matrix background. Of course, this is a partic-
ularly important problem if diluted samples have to be analyzed.
To our best knowledge, there were so far no attempts to establish
a more suitable matrix for cholesterol analysis. In particular it has
not yet been clarified, whether 9-AA represents a useful alternative
matrix: 9-AA normally detects only charged lipids such as phos-
pholipids, while an additional cationizing reagent (e.g. sodium ace-
tate) is required to generate ions from molecules that do not
possess charged functional groups. These aspects will be discussed
below in more detail and it will be outlined that the combination of
different matrices represents the method of choice to obtain a
complete data set [90].
458 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
There have been surprisingly few attempts to investigate cho-
lesteryl esters by MALDI MS [89,91]: in the same way as observed
for di- and triacylglycerols (see below), CEs are exclusively detect-
able as adducts with alkali metal ions (particularly sodium),
whereas the H+ adducts are not detectable at all. Although not
yet carefully investigated, this might be caused by the same reason
as described for triacylglycerols [92]: H+ adducts of cholesteryl es-
ters are less stable in comparison to the Na+ adducts and do not
‘‘survive” the flight distance to the mass analyzer of the MALDI de-
vice without fragmentation. The experimental fact that the MALDI
mass spectra of chromatographically pure CEs always exhibit a
small cholesterol peak is a strong confirmation of this mechanism.
3.6. Sphingolipids
Sphingolipids and glycosphingolipids are currently a hot re-
search topic because these compounds may be regarded to be
indicative of ageing and as disease markers. As there are some
recent reviews dealing with the subject of ‘‘sphingolipidomics”
[93,94], we will deal here only rather shortly with these com-
pounds but will discuss this topic in more detail in the context of
TLC/MALDI (see below). Here, we will focus primarily on the anal-
ysis of the most abundant sphingolipid, sphingomyelin (SM), the
structure of which is shown in Fig. 7.
SM is detectable in the same way as phospholipids and H+ as well
as Na+ adducts occur. Very recently a combination between MALDI
and ESI MS was used to study changes of the sphingolipid composi-
tion of parasitic nematodes [95]. It could also be proven that changes
of the lipid composition of tissues (and particularly of brain) often
affect much more the sphingolipids than the glycerophospholipids
[96]. Additionally, SM is an important constituent of virtually all
body fluids as well as tissues and, thus, particularly important in
the context of imaging studies of tissues [97]. Surprisingly, in com-
parison to our previous review [19], only a few papers dealing with
the MALDI MS analysis of sphingolipids appeared. Nevertheless, it
became obvious that a large variety of sphingolipids can be quanti-
tatively analyzed by MALDI-TOF MS in the presence of an internal
standard [98]: with sphingosylphosphorylcholine as the internal
standard, the relative peak heights of SM and ceramide monohexo-
side (CMH) could be used as a quantitative concentration measure
and linearity could be obtained between 50 and 1500 ng SM as well
as 5 and 150 ng CMH content, respectively. Nevertheless, despite
the similarities of the headgroups, SM is less sensitively detectable
in comparison to PC.
3.7. Glycerolipids
Triacylglycerols (where all hydroxyl groups of the glycerol are
esterified with fatty acids) are very abundant in animal organisms,
whereas compounds such as mono- or digalactosylglycerols are
particularly abundant in plants [99]. We will focus here primarily
on animals and only to a lesser extent on plant lipids. Therefore,
characteristics of TAGs (and to a minor extent diacylglycerols) will
be primarily discussed.
Fig. 7. Chemical structure of the backbone of sphingomyelin. ‘‘R” represents a
variable alkyl chain.
3.7.1. Di- and triacylglycerols
Triacylglycerols (TAG) are important for the storage of energy
(fat tissues in living organisms), while diacylglycerols (DAG) that
are normally generated from glycerophospholipids by cleavage of
the polar headgroup under the influence of the enzyme phospholi-
pase C are important lipid-derived second messengers [100]. The
positive ion MALDI-TOF mass spectra of both, DAGs [101] as well
as TAGs [102] can be easily recorded with standard DHB. The
MALDI mass spectra give always exclusively the Na+ adducts,
whereas H+ adducts are never detected, even if the solutions are
acidified [92]. Additionally, there are always intense fragment ions
corresponding to the loss of one sodiated fatty acyl residue. Using
MALDI MS in combination with high energy collisionally-induced
dissociation (CID), the losses of these fatty acyl residues can be
used for structural elucidation of TAGs [103], i.e. the determination
which fatty acyl residue is located in which position. It is important
to note that exclusively the Na+ adducts were useful for that pur-
pose while neither the K+ nor the Li+ adducts (that can be easily
obtained if the spectra are recorded in the presence of the corre-
sponding alkali salts) gave indicative fragment ion spectra [103].
The applied matrix has only a relatively weak impact on the
spectral quality, whereas strongly different sensitivities are
achieved in dependence on the used solvents. For instance, CHCA
and DHB dissolved in a mixture of acetonitrile and water gave only
sensitivities in the pmol range, whereas DHB in acetone provided
detection limits in the fmol range. Additionally, DHB provides a
weaker background than cinnamic acid derivates [104] that tend
to significant matrix cluster generation. This is even more impor-
tant than the achievable sensitivities because TAGs (e.g. from veg-
etable oils) are normally available in huge amounts. In order to
have highly reproducible spectra, sample preparation is very
important and particularly the ion content must be carefully con-
trolled. For instance, it was recently shown that 9-AA detects
TAG only in the presence but not in the absence of additional
cationizing reagents (‘‘dopants”) such as ammonium acetate [105].
It is somewhat surprising that independent of the used matrix,
DAG and TAG appear exclusively as the sodiated molecules,
whereas the H+ adducts are not detectable at all. A convincing
explanation has been recently given [92] and is based on the obser-
vation that the generation of fragment ions can be significantly re-
duced under alkaline conditions. Thus, it was suggested that the
observed fragments actually arise from unseen protonated TAGs
as their fragmentation occurs so rapidly and completely that pro-
tonated TAGs are normally not observed. If the pH is increased,
the H+ concentration is simultaneously decreased leading to re-
duced H+ adduct generation and, accordingly, to a lower yield of
fragmentation products [92]. Very recently it has been shown that
the yield of TAG fragments can be significantly reduced if the MAL-
DI target is covered with a thin nitrocellulose film prior to deposi-
tion of the TAG of interest [106]. The nitrocellulose film
simultaneously improves the shot-to-shot and sample-to-sample
reproducibility through the exhibition of a more homogeneous
matrix/analyte co-crystallization.
It has been repeatedly shown that MALDI MS is very suitable for
the screening of the compositions of crude TAG mixtures, for 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-
gation of the changes occurring in oil samples during frying [111].
This is a very important practical application because products
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
generated upon deep frying may include potentially hazardous
compounds [112]. Chromatographic enrichment of the polar com-
pounds prevented mass spectrometric ion suppression, thus allow-
ing the detection of minor species originating from thermal
oxidation. Particularly, diacylglycerols (DAG), oxidized TAG as well
as TAG dimers, and TAG fragments arising from the homolytic
beta-scission of linoleyl, peroxy, and alkoxy radicals were found
to be indicative of the frying process. Recently, lipid compositional
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
of mixtures.
3.7.2. Glycoglycerolipids
Plant and algal photosynthetic membranes contain relatively
small amounts of phospholipids, but high amounts of three glyco-
glycerolipids [99], monogalactosyl-diacylglycerol (MGDG), digalac-
tosyl-diacylglycerol (DGDG) and sulfoquinovosyldiacylglycerol
(SQDG). Due to differences in biosynthetic pathways, these lipids
contain high amounts of linoleic and, especially, alpha-linolenic
acids which have to be taken in the diet of most animals.
All glycoglycerolipids of plants can be easily characterized by
MALDI-TOF MS and some selected mass spectra are shown in
Fig. 8. Please note that the characteristic mass difference of
22 amu is not caused by H+/Na+ exchange, but both peaks corre-
spond to the Na+ adducts of lipids with different fatty acyl residues,
for instance DGDG 34:3 and DGDG 36:6. Therefore, it is unique of
these compounds – in the same manner as TAG and DAG that they
do never generate H+ adducts but exclusively Na+ adducts.
Please note that the discussed glycoglycerolipids can be easily
separated by means of TLC [69]. Therefore, the relative contribu-
tions of the individual lipid classes can be most easily determined
by TLC while the individual fatty acyl residues can be easily iden-
tified by MALDI-TOF MS. Finally, it has been shown by PSD MALDI
[114] that the fatty acyl residue in sn-1 position is preferentially
lost. This preferential loss may also be used for the determination
of the positions of the individual fatty acyl residues.
It has also been shown that MALDI MS combined with TLC is a
suitable, generally applicable method of lipid analysis [22].
3.8. Glycerophospholipids
Glycerophospholipids (GPL) are the most important subclass of
glycerolipids and merit, thus, a separate chapter. GPL are not only
of in vivo relevance as important constituents of the cellular mem-
branes, but some GPL (such as phosphatidic acid, phosphoinosi-
tides and lysophospholipids) are also involved in signal
transduction processes, i.e. they act as second messenger mole-
cules [115].
Therefore, GPL are attracting increasing interest as potential
disease markers in important, socioeconomically relevant diseases
such as atherosclerosis [116] or rheumatic diseases [117]. Many
different chromatographic, spectroscopic as well as MS methods
to assess the GPL composition of an unknown sample (e.g. an or-
ganic extract of a body fluid) are known and have been reviewed
[118,119]. The schematic structures of some selected GPL that
can normally be found in organic extracts from cells and body flu-
ids and that will be primarily discussed below in this review are
shown in Fig. 9.
Although ESI MS was so far primarily used in ‘‘lipidomics” stud-
ies [120], the number of lipid studies making use of MALDI MS is
increasing [121]. One potential reason for the nowadays extremely
growing interest in lipid analysis by MALDI MS is coming from
‘‘MALDI MS imaging” that is currently experiencing significant
interest [122] and will be explained below in more detail. As all rel-
evant tissues contain large numbers of cells and all cells possess a
459
membrane composed of considerable quantities of GPL and further
apolar components such as cholesterol, lipids are easily detectable
in all MALDI imaging experiments of tissues [123].
Despite the considerable technological progress that has been
achieved regarding the available MS hardware [124], aspects of
the ‘‘optimum” MALDI matrix (if it does anyway exist) have not
yet been clarified. Therefore, there is currently an increasing num-
ber of papers dealing with ‘‘matrix engineering” [21,29].
3.8.1. Zwitterionic glycerophospholipids (PC and PE)
Among all GPL so far investigated, phosphatidylcholine (PC) has
been by far, the most comprehensively studied. This has several
reasons:
1. PC is the most abundant constituent of cellular membranes – at
least as far animals or humans are concerned.
2. PCs are commercially available (for instance, from AVANTI Polar
Lipids, Alabaster, USA) with varying fatty acyl residues and,
thus, can be easily used as model compounds. Additionally,
alkyl- and alkenyl ether derivates are also available.
3. PC possesses a quaternary ammonia group that contains a per-
manent positive charge. Therefore, PC is most sensitively
detectable in lipid mixtures – at least if the positive-ion mode
is considered.
Regarding (3), i.e. the achievable sensitivity, it is important to
note that this is not only dependent on the MALDI device used
and, thus, the available mass analyzer and detector but also signif-
icantly on the applied matrix. Therefore, it is senseless to provide
detection limits without specifying the applied matrix. Since DHB
(2,5-dihydroxybenzoic acid) is one of the most established and
best characterized matrix compounds [125], the majority of the
available data were obtained with this matrix [126] and some se-
lected data are given in Table 1.
Table 1 clearly shows that different lipid classes exhibit very
different detection limits and points out that the charge state of
a lipid determines its detectability. Please note that sulfatides con-
taining a negatively charged sulfate group are much less sensi-
tively detectable than other lipids in the positive-ion mode.
Therefore, the comparison of the positive and the negative ion
mode is normally a straightforward approach and this is best done
by using different matrix compounds with different acidities, for
instance, DHB for positive ion and 9-AA for negative ion detection.
This will be explained below in more detail.
The significant importance of the matrix on the quality of MAL-
DI mass spectra of phospholipids was already recognized in a pio-
neering study by Harvey [127]. Although a large variety of different
matrices were tested, sinapinic acid, a-cyano-4-hydroxycinnamic
acid and DHB gave the best results regarding the achievable sensi-
tivity, resolution and the extent of (unwanted) fragmentation. It
was also noted that individual PL classes are detected with extre-
mely different sensitivities [127]. Particularly PE is much less sen-
sitively detectable than PC in the positive ion detection mode
(acidic PL such as PS are much more sensitively detectable as neg-
ative ions and will not be considered here): this is due to the per-
manent positive charge of the quaternary ammonia group of the PC
molecule, whereas the amino group of the PE can be easily depro-
tonated leaving the negative charge of the phosphate group [128].
This is the reason why the positive ion spectra of cell or tissue ex-
tracts are dominated by PC signals [121]. Therefore, separation into
the individual lipid classes is normally needed in order to analyze
the detailed lipid composition of mixtures. In contrast, the pre-
ferred detectability of certain lipid classes (particularly PC, LPC
and SM) may also be regarded as an advantage because the mass
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,
this low detection limit sheds light on the high sensitivities of
modern MS devices and particularly their detectors.
The high sensitivity for detecting PC is obviously an advantage if
derivatives of PC (i.e. with the same headgroup) are of interest. This
particularly concerns LPC that lacks in comparison to PC one fatty
acyl residue – normally in sn-2 position [135] – and the PC/LPC ra-
tio has been proven to be useful as disease marker for instance of
rheumatic diseases [117]. It has also been shown that LPC is not
only generated by the (most widely established) enzyme phospho-
lipase A2 (PLA2) but as well under the influence of reactive oxygen
species (ROS) such as HOCl [136]: in the first step of the reaction,
HOCl is added to one (or several) double bonds of the PC to gener-
ate the corresponding chlorohydrins. The characteristic mass shift
of 52 amu can be easily monitored by MS. The introduction of these
strongly electronegative atoms (chlorine and oxygen) leads to the
reduction of the strength of the ester bond making hydrolysis of
this bond easier and finally to the generation of LPC [137]. Thus,
LPC is obviously a marker of inflammatory conditions accompanied
by oxidative stress. Of course, in addition to HOCl there are many
more ROS that are generated under inflammatory conditions lead-
ing to different lipid oxidation products [138].
Although oxidation products such as endo- or hydro-peroxides
are also basically detectable by MALDI-TOF MS, they are only de-
tected with much lower intensities [139,140]. Even if the detailed
reasons are not yet known, it seems reasonable to assume that la-
ser irradiation leads (at least) to partial degradation of these ini-
tially formed products under generation of carbonyl compounds
upon cleavage of the original double bond. Very recently 6-aza-
2-thiothymine was suggested as the matrix of choice to detect oxi-
dized phospholipids [141]. A more detailed review dealing with
the mass spectrometric detection of oxidatively modified lipids is
available in [142].
Unfortunately, MALDI-TOF MS investigation of the products of
the reaction between HOCl and PE is much more difficult: PE con-
tains, in comparison to PC, an additional reactive functional group,
the amino group that exhibits a higher reactivity in comparison to
the olefinic residues. Therefore, the prime reaction products are
mono- as well as dichloroamines. It was shown that these products
can be detected by electrospray MS but not by MALDI-TOF MS be-
cause decomposition occurs rapidly [143]. However, this only
holds if standard matrices such as DHB or CHCA are used. In con-
trast, the expected products are easily detectable if a chlorinated
cinnamic acid (4-chloro-a-cyanocinnamic acid) derivate is used
[144] and the corresponding positive ion MALDI mass spectra are
shown in Fig. 12.
The underlying mechanisms for these surprising difference
were discussed in detail and related to the different proton affini-
ties of the individual matrices in [145]. This example proves clearly
that matrix design in order to develop more suitable matrices is
very important and not only of academic interest.
Finally, in addition to diacyl PLs there are also lipids with alke-
nyl ether bonds and these compounds are commonly termed
‘‘plasmalogens” [146]. Although these compounds can be analyzed
in exactly the same manner as common PCs, there is one important
difference: plasmalogens are extremely sensitive towards even
traces of acids that leads to the cleavage of the alkenyl ether link-
age under generation of the corresponding LPC. Although the acid-
ity of MALDI matrices such as DHB is tolerated, the use of TFA
(trifluoroacetic acid) as an cationizing agent is strongly discour-
aged because TFA in a concentration of about 0.1% is already suffi-
cient to induce partial hydrolysis of plasmalogens [147]. This is a
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
714.5
736.5
692.5
736.5
714.5
714.5
736.5
692.5
680 700 720
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
interest because these are characterized by high plasmalogen con-
tents [146].
3.8.2. Acidic glycerophospholipids
As expected, phospholipids such as PI, PS and PA are detectable
with rather poor sensitivities as positive ions due to their negative
charge [126]. Additionally, these lipids occur normally in rather
small amounts and, thus, they are rather difficult to detect. As they
are not commercially available to such an extent as PC and PE and
are often much more expensive, they have been much less often
investigated although they often have higher biological relevance
than the bulk phospholipids, PC and PE.
This particularly concerns phosphorylated phosphoinositides
(PPI) that possess three or more negative charges due to their addi-
tional phosphate residues [148]: all phosphoinositides produce
excellent signals in the negative ion mode when analyzed as iso-
lated compounds in amounts of about 1 nmol. However, the detec-
tion thresholds for phosphoinositides are higher than that of more
abundant PLs and increase further with the phosphorylation state.
While PC, for instance, is detectable in amounts of less than one
pmol, the amount of PIP3 on the sample plate that is needed to pro-
duce a sufficient signal is higher than 700 pmol [149]. Similar data
were also reported regarding the detection of cardiolipin [150] that
is also more highly charged than common PLs. Beside the charge, it
might also be possible that the increased polarities of these com-
pounds lead to reduced interactions with the matrix that is crucial
for the ionization process. However, comprehensive investigations
why phosphoinositides and cardiolipins are only scarcely detect-
able by MALDI MS are not available so far. This is a pity because
these compounds are particularly important, functional molecules.
Recently it has been shown [151] that PI species can be easily
monitored and even quantified in rather complex mixtures by
463
782.5
784.5
804.5 806.5
(c)
(b)
(a)
740 760 780 800 820
m/z[Th]
MALDI-TOF MS subsequent to the removal of interfering PC by
its binding to a small column filled with a silica gel cation exchan-
ger (SCX). A more comprehensive review of this important topic is
available in [152].
Although PS is a lipid that is of high diagnostic relevance partic-
ularly in the context of assessment of apoptotic cells [153], surpris-
ingly few attempts have been made to characterize PS by MALDI-
TOF MS. To our best knowledge there are just a very few papers
dealing with this topic: MALDI-TOF MS was recently used to inves-
tigate the lipid composition of tear samples from normal and dry
eyes of rabbits [154], whereby a solid ionic crystal MALDI matrix
of para-nitroaniline and butyric acid was used that gave superior
results in comparison to common crystalline matrices. In addition
to an enhanced SM content in the dry eye tears, PS species were
readily only detectable in dry eye tears but not in the normal tears.
Using artificial PL membranes, the influence of HOCl on PS was
investigated and many different products were detectable. As al-
ready discussed above, chlorinated compounds are only detectable
if 4-chloro-a-cyano-cinnamic acid is used as a matrix [155] but not
in the presence of standard matrices.
The detection of phosphatidic acid (PA) and derived lyso-phos-
phatidic acid (LPA) is a quite challenging task for the same reasons
as the phosphoinositides: PA and LPA possess a high negative
charge density and are, thus, much less sensitively detectable than
common zwitterionic phospholipids. Additionally, PA and LPA are
only present in very small amounts in biological samples. In order
to overcome these problems, an inorganic zinc complex ([1,3-
bis[bis(pyridin-2-ylmethyl)amino]propan-2-olato] dizinc(II)) was
used as matrix by Tanaka and coworkers [156] in order to trap
the LPA and to significantly enhance its detectability. 0.1 pmol of
LPA 18:1 was detectable on the MALDI target under these condi-
tions. This method was very recently improved and extended to
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-
cant clinical interest [157].
4. Analysis of lipid mixtures
The different detection limits of the individual PL classes as de-
scribed above and exemplarily reviewed in [126] are extremely
important for the analysis of biological samples: if crude organic ex-
tracts from cell cultures, body fluids or tissues are to be investigated
by MALDI-TOF MS without previous chromatographic purification,
it may happen – depending on the composition of the sample – that
only some selected lipid classes, particularly PL with quaternary
ammonia groups are detected while the remaining ones are sup-
pressed [158]. It is also a matter of fact that the different lipid classes
influence the intensities of others and it is often observed that
MALDI mass spectra change if they are recorded at different analyte
concentrations. This is illustrated with an extract from egg yolk as a
typical example (Fig. 13): it is evident that spectra change signifi-
cantly with increasing dilution and particularly the apolar compo-
nents, i.e. the triacylglycerols (TAG) are only detectable if diluted
solutions are investigated. Such suppression effects are well known
in MALDI-TOF MS and were thoroughly studied regarding the sup-
pression of matrix signals [159] as well as analyte signals [160].
Please also note that the ratio between the H+ (m/z = 760.6) and
the Na+ (m/z = 782.6) adduct of PC 16:0/18:1 is not constant but
the Na+ adduct is obviously favored with increasing dilution. A more
detailed discussion of these effects is beyond the scope of this paper,
but can be summarized as follows: when more analyte is present
than primary ions, suppression occurs as a consequence of second-
ary reactions and this effect is also significantly dependent on the
applied laser fluence. It is well known to each MALDI user that there
is a direct relation between signal intensity and analyte concentra-
tion only in a small concentration range, while the achievable signal
intensity decreases at higher concentrations. If the applied amount
of sample exceeds a certain limit, no signal is detectable anymore.
This behavior has been recently explicitly described [161]. There-
fore, it is highly recommended to use similarly concentrated solu-
tions of the analytes and to perform all measurements in the
presence of an internal standard.
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.
According to our experience there is no absolute need to use an
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,
or if a single compound (each for positive and negative ion detec-
tion) is sufficient. Although relative peak intensity ratios may also
be used, this method does not allow one to judge if both or only
one concentration are changing and, thus, the use of internal stan-
dards is recommended.
It is also highly recommended that the positive ion spectra are
recorded in the presence of an acidic (e.g. DHB) and the negative
ion spectra in the presence of an alkaline (e.g. 9-AA) matrix. If
DHB, the most common matrix in lipid analysis is used, PC and
other PLs with quaternary ammonia groups such as LPC or SM,
are detectable as positive ions, whereas negatively charged PLs
such as PI and PS are only detectable as negative ions or just
with very low sensitivity in the positive ion detection mode.
Using careful sample preparation, quantitative data regarding
the compositions of different cells [105], lipoproteins [89] as well
as meat [162] could be obtained. These are only some selected
examples but MALDI MS can surely be used to obtain quantita-
tive data. A more comprehensive survey of quantitative aspects
of MALDI MS regarding small molecule analysis is available in
[163].
There is another important aspect beside mass spectrometric
measurements: the influence of extraction on the lipid yield. Mix-
tures consisting of chloroform and methanol are the most estab-
lished and used in the majority of cases [118]. Although this
topic would be worth its own review, the following critical aspects
should be considered:
1. Enrichment of lipids by extraction with chloroform (according
to the methods developed by Bligh and Dyer [164] or Folch
et al. [165]) works quite well for bulk lipids such as PC or PE
and these lipids are nearly quantitatively extracted. However,
more polar compounds such as LPA or PPI are incompletely
extracted because these compounds have a higher affinity to
remain in the water/methanol phase. If these molecules are of
prime interest more polar solvent mixtures and/or acidifying
the solution is required.
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
2. Surprisingly, apolar lipids are also incompletely extracted if
standard methods such as the Bligh and Dyer approach [164]
are used and more apolar conditions should be used for that
purpose. The reason for this incomplete extraction is most
probably the tendency of apolar lipids to form other structures
than amphiphilic PL that provide well-defined supramolecular
structures.
3. If an aqueous sample containing proteins is treated with
organic solvents, precipitation of proteins and other highly
polar compounds will occur. This is obvious by the generation
of a white ring at the interface between the aqueous and the
organic phase. Some lipids may stick to the precipitated pro-
teins and are, thus, lost if only the organic layer is used for fur-
ther analysis.
In order to make a long story short: there is obviously not a sin-
gle method that allows the unequivocal extraction of all PL classes
and great care is required if absolute, quantitative data are re-
quired. There was one 31P NMR based study that has compared
the use of detergents and organic solvents for the (lipid) extraction
of human erythrocytes [166]. It was one result of this study that
extraction with the detergent sodium cholate in comparison to or-
ganic solvents resulted in a much higher yield of PLs and would,
thus, be the method of choice in order to extract lipids quantita-
tively. Unfortunately, this method is only applicable to 31P NMR
where the presence of the detergents does not play a role because
they are not detected at all. In contrast, interference has been
shown recently following the treatment of boar spermatozoa with
different detergents and subsequently assessing the lipid composi-
tions of the extracts by MALDI-TOF MS: in the presence of even
small amounts of detergents, the sensitivities for lipid detection
were strongly reduced [167]. Thus, the potential presence of deter-
gents is a considerable problem: detergent removal kits are avail-
able for proteins, but there is, so far, no commercial detergent
removal kit for the organic (lipid containing) phase. This is caused
(a) by the lower interest that lipids were so far experiencing and
(b) the fact that most detergents exhibit a significant distribution
of their molecular weights. This makes their removal much more
difficult in comparison to compounds with a defined molecular
weight. In order to illustrate this fact in Fig. 14 the positive ion
MALDI-TOF mass spectrum of a commercially available TWEEN
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.
465
20 preparation is shown: although we do not want to discuss this
spectrum in detail (further details are available in [167,168]), it is
evident that the spectrum of this detergent is very complex and
covers a significant mass range. This makes detergent removal
rather difficult and, therefore, the application of detergents to ex-
tract lipids from biological sources seems not to be the method
of choice. Nevertheless, there are some papers dealing with this ap-
proach and the use of dodecyl maltoside for membrane solubiliza-
tion seems preferential because this detergent exhibits a lower
extent of heterogeneity [169]. The reader should also note that
parameters such as the salt concentration influence the achievable
spectral quality although this is a much smaller problem in the
case of lipids because the majority of the salt is already removed
by the extraction process [170]. Nevertheless, in order to account
for different salt concentrations, all adducts (normally the H+ and
Na+ adduct) of a selected lipid must be used for subsequent data
analysis. This means that the combined intensities of both adducts
must be used for proper quantitative analysis.
Regarding mixture analysis, one should also wonder if complete
information is really always needed. Would less sometimes be
more? This particularly holds because the difficulties of proper
data analysis often strongly increase as more data are collected.
According to our opinion it is often sufficient to have some defined
selected metabolites determined. For instance, we have established
the PC/LPC ratio as a measure of the quality of a human ejaculate
[171] that correlates very well with the amount of annexin-V-po-
sitive spermatozoa. Very recently, we have also shown that this
is not only possible for human spermatozoa but may also be ap-
plied to the analysis of animal spermatozoa, for instance from cats
and cattle [172]. The structures of the related molecules are shown
in Fig. 15. Although it is not yet clear whether (a) increased activity
of PLA2 or (b) an enhanced generation of ROS or (c) a reduced
reacylation of the LPL primarily contribute to the changed PC/LPC
ratio, this ratio seems a suitable measure of the quality of sperma-
tozoa. A very simple method to quantitatively extract LPC from
aqueous samples that uses only methanol and necessitates only a
single centrifugation step was suggested recently [173].
Somewhat related to spermatozoa research, it was very recently
shown that different animal oocytes provide very different MALDI
mass spectra and can be, thus, easily differentiated [174] and give
molecular fingerprints of the lipid composition.
466 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
5. Separation of the individual lipid classes prior to analysis
When separated, all phospholipid classes (if present in suffi-
cient amounts) are easily detectable by MALDI MS because sup-
pression effects between the individual lipid species do not play
any role. Nevertheless, detection limits for the individual lipid clas-
ses [126] are very different due to the reasons discussed above.
Separation into the different lipid classes may be of course per-
formed by offline HPLC and the fractions obtained subsequently
analyzed by MALDI-TOF MS. We will not pay major attention to
this application because HPLC is normally not coupled to a MALDI
but to an electrospray ion source and this important method of li-
pid analysis has been already comprehensively reviewed recently
[175]. This also holds for the use of, for instance, APCI instead of
ESI [176].
In contrast, we will focus here on thin-layer chromatography
(TLC), that is even nowadays still a widely used separation method
in lipid research [177] due to its simple and fast (several samples
can be separated at the same time) performance as well as the very
moderate price of the required equipment. TLC is also very ecolog-
ically-friendly because much lower amounts of solvents are
needed in comparison to HPLC. Finally, certification of TLC separa-
tions is simpler in comparison to HPLC: using TLC, a completely
new stationary phase is always used, whereby potential contami-
nations from a previous run can be surely avoided, while in the
context of HPLC one may always suspect that there is some resid-
ual amount of the analyte even subsequent to intense washing of
the column [178].
Clearly, TLC separation may be also offline coupled to MALDI:
the lipid mixture of interest is separated by TLC and afterwards
stained by a non-destructive dye that does not lead to alterations
of the masses of the lipids of interest. This requirement is fulfilled,
for instance, by the fluorescent dye primuline that binds non-cova-
lently to the fatty acyl residues of lipids [179] and can be easily re-
Fig. 15. Survey of the conversion of PC into LPC under the influence of the enzyme PLA2 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.
moved by changing the polarity of the solvents. Afterwards the
silica gel with the attached lipid is scraped off from the TLC plate,
the lipid is eluted by organic solvents and subsequently analyzed
by MALDI MS. Some selected examples of this technique are the
analysis of characteristic lipid oxidation products [180], cells such
as bull [147] or human spermatozoa [181], body fluids such as lung
surfactant [182], tissues such as brain [183] or organic extracts
from algae [69].
Although this approach is capable of providing convincing re-
sults, it is obviously boring and time-consuming, particularly if
many samples each containing a lot of different lipid species have
to be analyzed. Additionally, this method bears the risk of losing
certain lipid species. For instance, it has been shown that an ‘‘effect
of chromatography” exists [184]. This means that lipids with dif-
ferent fatty acyl residues are differently tightly bound to the silica
gel and are, thus, eluted to a different extent. Therefore, the fatty
acyl compositions monitored prior and subsequent to chromatog-
raphy may be different [184].
Despite these problems, a method enabling a more direct cou-
pling between TLC and ESI MS was recently successfully estab-
lished [185]. A plunger based extraction interface (now
commercially available as the ‘‘ChromXtract” from the CAMAG
company) combined with an HPLC pump was shown to provide
good results for quantitative TLC/ESI MS from HPTLC silica gel
plates regarding repeatability of the MS spectra and the achievable
limit of detection (LOD) and the limit of quantification (LOQ). This
was shown using harmane, a heterocyclic aromatic amine, as the
selected model compound [186]. Modifications of the device
enabled complete, highly reproducible extraction of analytes
from glass backed as well as aluminum backed TLC and HPTLC
plates, layers with thicknesses up to 100 lm and different station-
ary phases [187]. This application is surely a current ‘‘hot” topic
[188,189] and of interest for scientists working in different
fields.
 B. Fuchs et al. / Progress in Lipid Research 49 (2010) 450–475
Since a ‘‘solid” sample is used in TLC, i.e. the lipid is dispersed in
a solid silica gel ‘‘matrix”, the idea to combine MALDI directly with
TLC seems a straightforward approach and was already attempted
at the end of the 1990s [190]. MALDI characterization of the devel-
oped TLC plate would offer the advantage of much higher resolu-
tion because the achievable resolution is determined primarily
by the laser spot size that is normally of the order of only about
20 lm. This means that 50 individual MALDI mass spectra can be
recorded from a TLC spot of a diameter of 1 mm and provides the
possibility to resolve different components that could never be re-
solved if the complete spot is scraped from the TLC plate. However,
there are three important problems that must be solved [22]:
1. The matrix must be supplied in a very homogeneous fashion
and in the form of very small droplets since otherwise the
achievable resolution would be compromised by the spreading
of the matrix solution on the TLC plate as well as the formation
of large matrix–analyte crystals.
2. The majority of commercially available MALDI-TOF devices are
currently equipped with UV lasers (k = 337 nm). UV irradiation
does not penetrate very deeply into the silica layer and, thus,
primarily compounds near to the surface of the TLC plate are
detected. Due to these problems, quantitative aspects have
not yet been comprehensively addressed. This is a particular
problem regarding the fact that different compounds are
located at different silica gel depths in dependence on their
RF-(retardation factor)-values. Therefore, IR-MALDI seems a
suitable wayout because the IR radiation penetrates much dee-
per into the silica gel than UV.
3. The achievable mass spectrometric resolution as well as mass
accuracy is surely much lower if spectra are recorded directly
from a TLC plate in comparison to a standard metal MALDI tar-
get. However, this is a minor problem in the field of lipid anal-
ysis (in comparison to, e.g. tryptic digests of proteins) because
the smallest mass difference within a certain lipid class that
must be still resolved by MS is 2 amu, corresponding to one
double bond.
5.1. Glyco- and sphingolipids
The majority of applications of TLC/MALDI have so far been ded-
icated to the analysis of glycolipids, while phospholipids have been
investigated to a much lesser extent [22].
In a rather early attempt [191], native glycosphingolipids were
separated on a conventional TLC plate and subsequently heat-
transferred to a different membrane consisting of polyvinylidene
difluoride (PVDF) where the MALDI measurements were per-
formed. This was done in order to remove impurities that are
potentially coming from the TLC material and because spectral
quality and particularly the achievable sensitivity can be highly
improved under these conditions.
In 2004, it could be shown that the analysis of gangliosides is
possible by direct TLC/MALDI without major fragmentations of
the analyte [192]. In order to enhance the stability of the generated
ions, these authors used a relatively high gas pressure to allow col-
lisional ‘‘cooling” of the generated ions [192]. Recently is was also
shown that glycolipids from brain can be analyzed by using a very
simple equipment, i.e. a commercially available MALDI-TOF MS de-
vice equipped with a standard nitrogen laser [193]. The majority of
TLC/MALDI studies of glycolipids have used UV lasers. However,
there are also some studies where infrared lasers were applied.
These are primarily available on homebuilt MALDI devices but
commercially only on special request. Er:YAG (Erbium-doped yt-
trium aluminum-Garret) IR lasers are normally used and have
the considerable advantage that IR radiation penetrates deeper
into the sample than UV radiation. Therefore, bringing the com-
467
plete analyte from the inner of the plate to the TLC surface is less
important. Beside an IR laser, Dreisewerd [194] and coworkers
used an orthogonal but not an axial system for glycolipid analysis.
The orthogonal configuration has the significant advantage that
irregularities of the surface of the sample do not reduce the achiev-
able mass accuracy. It was found that even minor gangliosides
from a complex lipid mixture extracted from cultured Chinese
hamster ovary cells can be identified.
These authors [194] provided also convincing evidence that the
fluorescent dye ‘‘primuline” that is widely used in lipid research as
it binds non-covalently to the fatty acyl residues [179] may be used
as a non-destructive, TLC/MALDI-compatible staining agent. Similar
data were independently obtained by another group that addition-
ally introduced ‘‘vibrational” cooling [195]. The term ‘‘vibrational
cooling” describes the desorption process in the pressure range,
where ‘‘cooling” of the excess energy of the generated ions is
achieved. Under these conditions, fragmentation of labile analytes
can be minimized. This is normally performed by introducing an in-
ert gas (e.g. N2) under a moderate pressure into the MS device.
In a recent study 2,5-dihydroxybenzoic acid (DHB) in acetoni-
trile/water (1:1, v/v) was found very useful as matrix for the anal-
ysis of glycosphingolipids because in this solvent mixture DHB is
highly soluble and this solvent mixture has a relatively high sur-
face tension leading to reduced analyte spreading. Sensitivities be-
tween 25 and 50 pmol could be obtained under these conditions. It
should be noted that the application of highly concentrated matrix
solutions is very important because a larger excess of matrix over
the analyte in comparison to standard MALDI (i.e. the use of a
stainless steel target) is required for direct TLC/MALDI coupling
in order to minimize fragmentation of the analyte [22].
A combination between MALDI, TLC and antibody detection was
also recently suggested [196,197]: these authors used the follow-
ing workflow: (a) TLC separation of cancer-associated glyco-
sphingolipids (GSLs) from human hepatocellular and pancreatic
tumors (b) their detection with oligosaccharide specific proteins
and (c) the direct in situ MS analysis of the GSLs previously de-
tected by the antibody. Detection limits of less than 1 ng of immu-
nostained GSLs could be obtained. It is a particular advantage of
this approach that crude lipid extracts of biological origin can be
directly used for TLC-IR-MALDI-MS and no laborious, previous
GSL purification is needed.
A ‘‘TLC-Blot-MALDI-Imaging” method was recently also sug-
gested [198] to visualize individual molecular lipid species. This
method was indicated to have higher sensitivity than common
staining methods and allows for direct visualization of all relevant
lipids within a linear range of approximately one order of magni-
tude. The important field of glycolipid analysis – particularly by
combined TLC/MALDI has been recently comprehensively re-
viewed [199].
One important problem that is not only of relevance to TLC/
MALDI but as well to MS imaging of biological tissues stems from
the interference of matrix peaks with the signals of the molecules
of interest. In order to overcome this problem, it was suggested to
use graphite-assisted laser desorption/ionization (GALDI) MS and
this technique has already been successfully applied to the analysis
of cerebrosides in a total brain lipid extract of high complexity
[200]. Beside the lack of typical ‘‘matrix” peaks it could also be
shown that GALDI suffers far less from suppression effects, for in-
stance, the suppression of cerebrosides by other dominant lipid
species, particularly PC.
In addition to positive ion detection, (negatively charged) sulfa-
tides could be also easily detected using a thin layer of graphite.
Thus, graphite does not only enable the recording of positive but
also of negative ion spectra although details of the basic ionization
have to be elucidated. So far, however, 9-AA seems to be the matrix
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
into the TLC plate, but simultaneously confers the disadvantage
that abundant glycerol adducts (and to a minor extent even NaCl
adducts) of the PLs of interest are detected and complicate inter-
pretation of the spectra. Another problem is that IR lasers are com-
mercially barely available so far. Therefore, another approach used
a readily available N2 laser and standard DHB as matrix [202].
One selected lane of a TLC-separated hen egg yolk extract and
some selected MALDI mass spectra are shown in Fig. 16.
Two facts are particularly remarkable in the context of Fig. 16.
First, even rather minor fractions of, e.g. phosphatidylinositol (PI)
can be easily analyzed and give spectra with a convincing signal-
to-noise (S/N) ratio. As PI makes out only about 0.5% of the total
PLs of egg yolk [202], this clearly proves the significant dynamic
range of more than two orders of magnitude. Second, the lower
and the upper part of the PE spot provide significantly different
mass spectra, whereby PEs with longer fatty acyl (in particular
arachidonoyl) residues are found in the upper and PEs with shorter
acyl residues in the lower part of the spot. This is a clear indication
that even the separation quality obtained under normal phase con-
ditions is sufficient to allow the differentiation of fatty acyl resi-
dues if MS is used for subsequent analysis [203].
Regarding detection limits, both approaches [150,202] provided
comparable results (about 400 pmol for PC detection) and, there-
fore, both approaches might be useful for routine lipid analysis.
This particularly holds as surprisingly good mass accuracies (about
100 ppm) and mass resolutions (about 3000) could be obtained
that are absolutely sufficient for the differentiation of individual
PL species.
Very recently, an automatic routine for the spatially-resolved
screening of a developed TLC plate became available and was al-
ready successfully applied for the investigation of the lipid compo-
sition of human mesenchymal stem cells [204] as well as plasma
PL signatures associated with respiratory disease severity in cystic
Fig. 17. Positive-ion MALDI-TOF mass spectrometric image (left) and densitometric
image (right) of a developed HPTLC plate containing lipids from hens’ egg yolk.
Assignments of the individual spots and the m/z values of the signals that were used
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
be easily differentiated form those with shorter fatty acyl residues. Reprinted with
permission from Journal of Planar Chromatography 22 (2009) 35–42 [203].
469
fibrosis patients [205]. This is a clear indication that TLC/MALDI
data can be basically quantitatively analyzed although further at-
tempts are necessary to establish more useful and sensitive matrix
compounds – particularly for negative ion detection directly on the
TLC plate [131,206].
Due to the much higher experimental time that is required to
record an MS image, 1D slices are normally investigated in the case
of a developed TLC plate. However, 2D MS images can be also easily
acquired and this is shown in Fig. 17.
MALDI MS imaging, a fast developing application of MS will be
discussed more comprehensively in the following section.
6. MALDI MS imaging
Microscopic methods and radiological investigation techniques
(computer tomography, magnetic resonance imaging and sonogra-
phy) are well-known imaging methods. These methods have been
established for several decades, whereas MS imaging is a rather
new, but very promising approach – not only in the basic sciences
but as well in clinical diagnosis [207]. Although there are already
other MS imaging methods based on desorption electrospray
(DESI) MS [208] or secondary ion mass spectrometry (SIMS)
[209] available (for a timely review see [210]), we will focus here
exclusively to applications of MALDI MS. However it should be
noted that DESI MS has clear advantages if the analysis of smaller
lipids is of interest. For instance, it could be convincingly demon-
strated upon the DESI image analysis of thin tissue sections of 68
samples of human prostate cancer and normal tissues that choles-
terol sulfate (m/z = 465) is a useful marker of prostate cancer [211].
Although common (commercially available) UV lasers penetrate
only a few lm into a solid sample such as thin tissue slices [20],
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.
3. The achievable image quality is significantly dependent on the
sample preparation and particularly on the even spotting of
the tissue slice with the matrix. Already small inhomogeneities
of the matrix deposition will seriously compromise the image
quality. Of course, this also holds for slight fluctuations of the
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
context of successful MALDI MS imaging were recently summa-
rized by Bernhard Spengler, one of the pioneers in this field [212]
in [20]. Therefore, we do not want to give so many methodological
details but will focus on some selected applications.
The simplest approach to perform MALDI MS imaging is to fix
the tissue slice onto the MALDI target and to cover it with matrix.
The homogeneous spotting of the matrix is unequivocally the most
critical point and many efforts for its improvements were and still
are undertaken [213] and devices for homogeneous matrix deposi-
tion onto the sample of interest by sophisticated spraying tech-
niques are nowadays commercially available.
Although not originally in the focus of interest, different classes
of lipids are primarily detected if native tissue slices are investi-
gated by MS imaging because these species ionize particularly well
and lipids are abundant constituents of all tissues. Thus, a much
higher interest in lipid analysis by MALDI-TOF MS has been obvi-
ous over the last five years [214]. This particularly holds because
there is increasing evidence that not only proteins but also certain
lipid classes are useful as disease markers. For instance, it has re-
cently been suggested that LPC may be used as a marker molecule
of ovarian cancer [215] and that differently saturated LPCs possess
either inflammatory or anti-inflammatory properties [216]. A typ-
ical example of a positive ion MALDI image of a mouse brain is
shown in Fig. 18
Fig. 18 shows the type of high quality images that can be gen-
erated for individual lipid species in tissues using MALDI MS, in
which clear anatomical distribution of these species are observed.
This figure compares an optical image of a mouse brain sagittal
section with three 2D ion intensities maps generated by MALDI
imaging. The MALDI images were recorded in positive-ion mode
and sublimation of the matrix was employed to coat the tissue
[213]. In addition to brain (that is often investigated because this
tissue is particularly rich in a large variety of lipids [193]) the lipid
compositions of many different tissues have already been success-
fully investigated by MALDI-TOF MS imaging [217] and these
compositional data compared with results from established bio-
chemical methods. A surprisingly good agreement between both
methods is often found.
Therefore, tissue samples can be analyzed for their lipid compo-
sitions by direct MALDI-TOF MS without sample workup [218].
It seems that the application of liquid ionic matrices provides
superior image qualities in comparison to conventional crystalline
matrices due to the more even distribution of the matrix on the tis-
sue surface [219]. This is the most important prerequisite for using
MALDI-TOF MS as a (quantitative) imaging technique [220], i.e. to
obtain spatially-resolved information about the PL distribution
within a given sample. Unfortunately, the achievable resolution
is normally not sufficient to obtain images with cellular resolutions
[20]. Thus, the combination between MALDI and SIMS imaging is a
very promising approach: while SIMS is capable of providing
images with a subcellular resolution, MALDI is unequivocally the
method of choice to detect lipids with relatively high masses such
as cardiolipins [221].
Although the lipid compositions of many different tissues (e.g.
brain [218], eye lenses [220], and dystrophic muscle [222]) have al-
ready been examined by MALDI-TOF MS, the most intriguing suc-
cess of MALDI imaging was to prove that it is possible to
differentiate tumor tissue from the ischemic and necrotic areas
of the lesion [223]. Already this selected example clearly indicates
that MALDI imaging will have great future developments because
it turns increasingly out to be useful for clinical diagnostics. A com-
prehensive review dealing with MS imaging - not only of the re-
lated lipid aspects - is available in [122].

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
7. Summary
Unequivocally there is considerable interest in lipid analysis
and it is expected that this interest will increase in the future be-
cause many diseases are recognized to be accompanied by altera-
tions in the lipid compositions of either body fluids or tissues or
even both. Thus, lipids are of unequivocal diagnostic relevance.
Many different methods of lipid analysis are nowadays established
and among these methods mass spectrometric techniques
unequivocally have the most significant potential [224]. The choice
of the most suitable method does not only depend on the suitabil-
ity of the method to solve the problems of interest but also on the
availability of the different techniques. Since many MALDI mass
spectrometers were purchased in the context of proteomics and
genomics initiatives, it was one major aim of this review to provide
evidence that these devices are not only useful in the field of polar
biomolecules but also for apolar ones, such as lipids: although not
yet commonly accepted, MALDI-TOF MS represents a reliable
method of lipid analysis. Hopefully, we were able to show in this
review that basically all lipid classes can be analyzed by this meth-
od - although some lipid classes are more refractive to analysis
than other ones. This particularly concerns relatively large lipids
(cardiolipins), highly polar lipids (poly-phosphoinositides) and sul-
fatides that tend to loose the sulfate residue. Here further method-
ological developments - particularly in the field of (MALDI) matrix
engineering - are required.
Nevertheless, MALDI MS has several important advantages:
measurements can be performed in a short time and a very conve-
nient way. Only minor, less time-consuming sample purification is
required because considerable amounts of impurities such as salts
are tolerated. Therefore, MALDI-TOF MS is useful for the routine
analysis of a large number of samples. As soon as a suitable device
is available, MALDI analyses may also be regarded as relatively
inexpensive in comparison to biochemical assays.
The interest in MALDI lipid analysis has been significantly in-
creased with the establishment of MS imaging techniques during
the past decade: as lipids are abundant in tissues due to the high
number of cells and lipids ionize also particularly well, lipids are pri-
marily detected under MS imaging conditions. As the spectral inten-
sities are used to create the image, this is a strong indication that
quantitative analysis of lipid concentrations by MALDI-TOF MS is
possible. However, only certain lipid classes are detectable under
these conditions because individual phospholipid classes are char-
acterized by different peak intensities: the concentration of com-
pounds with quaternary ammonia groups such as PC or SM is
overestimated, whereas the concentration of other lipids is underes-
timated. This holds at least if positive ion MALDI mass spectra are re-
corded. Using additionally negative ion detection and/or different
(for instance 9-AA and DHB) matrix compounds, this problem may
be at least partially overcome and it is assumed that different mark-
ers of the different lipid classes will become detectable.
However, for the detailed analysis of complex mixtures, previous
separation of the sample into the individual lipid classes is required
– in the same manner as if LC/MS would be used. For this purpose,
combined TLC/MALDI may be used because the sample dispersed
in a solid matrix (the silica gel) can be easily combined with a MALDI
ion source. Therefore, the same possibilities are available as in the
case of a LC/MS system. The future will show whether MALDI-TOF
MS will become a method of similar importance as the so far estab-
lished LC/MS methods in the field of lipid analysis.
Acknowledgments
The authors wish to thank all colleagues and friends who
helped them in writing this review. Particularly the kind and help-
471
ful advice of Dr. Suckau and Dr. Schürenberg (Bruker Daltonics,
Bremen) as well as Dr. Thorsten Jaskolla (Goethe University, Frank-
furt) is gratefully acknowledged.
This work was supported by the German Research Council (DFG
Schi 476/12-1 and FU 771/1-1 as well as TR 67 project A2) and the
Federal Ministry of Education and Research of the Federal Republic
of Germany (‘‘The Virtual Liver”, 0315735).
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