2,5-Dihydroxybenzoic acid salts for matrix-assisted laser desorption/ionization time-of-flight mass spectrometric lipid analysis: Simplified spectra interpretation and insights into gas-phase fragmentation

Article abstract of DOI:10.1002/rcm.6910

RATIONALE: In the last decades the interest in lipids as important components of membranes has considerably increased. Nowadays, lipids are often routinely analyzed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). In this regard, many relevant aspects are so far unknown, e.g., gas-phase stabilities, adduct formation and fragmentation. To fill this gap, MALDI matrix salts are presented which allow for simplified lipid analysis and elucidation of the underlying gas-phase fragmentation mechanisms. METHODS: MALDI-TOF MS was used due to its beneficial properties for lipid investigations, e.g., high sensitivity, simple sample preparations, and a high tolerance to contaminants. The lipid hydrolysis, ionization and fragmentation properties of synthesized near neutral Na⁺ and NH ₄⁺ salts of the commonly used MALDI matrix 2,5-dihydroxybenzoic acid were compared to that of DHB free acid itself as well as to base addition to DHB during dried-droplet sample preparation. RESULTS: Many lipid classes such as sterols, triacylglycerols, phosphatidylcholines and -ethanolamines undergo initial protonation with subsequent prompt partial up to quantitative fragmentation when analyzed with classical acidic matrices by MALDI-TOF MS. Neutral matrix salts can prevent initial analyte fragmentation by suppression of analyte protonation. Additionally, intramolecular gas-phase fragmentation reactions can be inhibited due to analyte stabilization by cation chelation. Base addition during sample preparation leads not only to in situ generation of matrix salts but also to analyte hydrolysis. CONCLUSIONS: Neutral DHB salts avoid separation of lipid species into several ionization states when used as matrices in MALDI-TOF MS. This allows for simplified lipid spectra interpretation. Due to the high cationization efficiency of DHB matrix salts, certain lipid classes become detectable which cannot be analyzed easily using standard acidic DHB. Copyright

Full text of DOI:10.1002/rcm.6910

Research Article
Received: 16 January 2014
Revised: 25 March 2014
Accepted: 30 March 2014
Published online in Wiley Online Library
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363
(wileyonlinelibrary.com) DOI: 10.1002/rcm.6910
2
,5-Dihydroxybenzoic acid salts for matrix-assisted laser
desorption/ionization time-of-flight mass spectrometric lipid
analysis: Simplified spectra interpretation and insights into gas-
phase fragmentation
1
2
3
Thorsten W. Jaskolla *, Kristin Onischke and Jürgen Schiller
1
Institute of Hygiene, University of Münster, Robert-Koch-Str. 41, 48149 Münster, Germany
Merz Pharma GmbH & Co. KGaA, Am Pharmapark, 06861 Dessau-Roßlau, Germany
Institute of Medical Physics and Biophysics, Faculty of Medicine, University of Leipzig, Härtelstr. 16-18, 04107 Leipzig, Germany
2
3
RATIONALE: In the last decades the interest in lipids as important components of membranes has considerably
increased. Nowadays, lipids are often routinely analyzed by matrix-assisted laser desorption/ionization time-of-flight
mass spectrometry (MALDI-TOF MS). In this regard, many relevant aspects are so far unknown, e.g., gas-phase
stabilities, adduct formation and fragmentation. To fill this gap, MALDI matrix salts are presented which allow for
simplified lipid analysis and elucidation of the underlying gas-phase fragmentation mechanisms.
METHODS: MALDI-TOF MS was used due to its beneficial properties for lipid investigations, e.g., high sensitivity,
simple sample preparations, and a high tolerance to contaminants. The lipid hydrolysis, ionization and fragmentation
properties of synthesized near neutral Na+ and NH4+ salts of the commonly used MALDI matrix 2,5-
dihydroxybenzoic acid were compared to that of DHB free acid itself as well as to base addition to DHB during dried-
droplet sample preparation.
RESULTS: Many lipid classes such as sterols, triacylglycerols, phosphatidylcholines and -ethanolamines undergo initial
protonation with subsequent prompt partial up to quantitative fragmentation when analyzed with classical acidic
matrices by MALDI-TOF MS. Neutral matrix salts can prevent initial analyte fragmentation by suppression of analyte
protonation. Additionally, intramolecular gas-phase fragmentation reactions can be inhibited due to analyte stabilization
by cation chelation. Base addition during sample preparation leads not only to in situ generation of matrix salts but also
to analyte hydrolysis.
CONCLUSIONS: Neutral DHB salts avoid separation of lipid species into several ionization states when used as matrices
in MALDI-TOF MS. This allows for simplified lipid spectra interpretation. Due to the high cationization efficiency of
DHB matrix salts, certain lipid classes become detectable which cannot be analyzed easily using standard acidic DHB.
Copyright © 2014 John Wiley & Sons, Ltd.
The interest in lipid research has considerably increased during
the last decades due to the discovery of the physiological
structural component of cellular membranes and massively
involved in the formation of liquid ordered membrane
domains. Cholesteryl esters (as one important constituent of
[1]
[3]
importance of lipids, for instance, as second messengers,
disease markers, or important (bio)membrane constituents.
[
2]
[3]
lipoproteins) are the transporters of fatty acids in the organism.
Nowadays, many different chromatographic and
spectroscopic methods of lipid analysis are established. Mass
spectrometric methods, for instance electrospray ionization
Phospholipids (PLs) are characterized by a polar headgroup
and two apolar fatty acyl residues linked by a glycerol
backbone. Therefore, PLs exhibit amphiphilic properties and
form typical lipid bilayer structures in an aqueous
[5]
[6]
(ESI) or atmospheric pressure chemical ionization (APCI),
[4]
environment. The most abundant PLs in the majority of
biological systems are phosphatidylcholines (PCs) and
phosphatidylethanolamines (PEs). Further important lipids
are triacylglycerols (TAGs) which are essential for nutrition as
well as energy storage and cholesterol which is an important
play particularly important roles regarding the analysis of
lipids due to their capacities to detect the almost entire diversity
of naturally occurring lipid species in combination with
unmatched sensitivities. Matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry (MALDI-TOF MS)
[7]
is also increasingly used for lipid analysis because of its
simple and fast performance, robustness against impurities
and generally high sensitivity. Standard MALDI-TOF mass
spectra (i.e., without sophisticated sample purification) of
physiological (phospho)lipid samples are normally dominated
by the simultaneous presence of proton and sodium adducts
*
Correspondence to: T. W. Jaskolla, Institute of Hygiene,
Biomedical Mass Spectrometry, University of Münster,
Robert-Koch-Str. 41, 48149 Münster, Germany.
E-mail: TJaskolla@uni-muenster.de
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363
Copyright © 2014 John Wiley & Sons, Ltd.

T. W. Jaskolla, K. Onischke and J. Schiller
[
8]
of the individual lipids in the positive ion mode. Division of a
lipid into more than one detectable species can hardly be
avoided but is undesirable since the sensitivity is reduced and
the presence of different adducts can complicate unequivocal
Na
2
CO
3
decahydrate (928.30 mg; MW= 286.14 g/mol,
3.24 mmol) and heated under stirring for 20 min until CO
2
evolution ceased and a clear pH-neutral solution was obtained.
The hot solution was concentrated to dryness. The DHB
sodium salt (NaDHB) was pre-dried at 50 °C with subsequent
drying at reduced pressure until constant mass.
[
9,10]
peak assignments.
However, in some cases the diversity is
reduced by the absence of protonated lipid species: for instance,
TAGs never occur as protonated but exclusively as cationized
[11]
(
typically sodiated) ions in MALDI-TOF mass spectra.
DHB ammonium salt – ammonium 2,5-dihydroxybenzoate
Nevertheless, it has been assumed that TAGs can indeed be
initially generated as protonated species but undergo prompt
and nearly quantitative fragmentation yielding neutral fatty
Ammonium bicarbonate (513 mg; 1 equiv.) suspended in
MeOH (5 mL) was slowly added to DHB (1 g; 1 equiv.;
6.48 mmol) dissolved in MeOH (2 mL) under stirring. The
[12]
acids and positively charged diacylglycerol (DAG)-like ions.
mixture was stirred until all NH
generation of dissolved DHB ammonium salt (NH
was complete when the solid ammonium bicarbonate
disappeared and the CO evolution ceased. The clear yellow
4
HCO
3
reacted to CO
2
. The
In agreement with this, Gidden et al. reported that the yield of
DAG-like ions can be significantly reduced under strongly
alkaline preparation conditions (1 M NaOH or 14 M NH3)
which seem to prevent the initial generation of unstable
4
DHB)
2
[
13]
solution was evaporated using a vacuum evaporator and
pre-dried at 50 °C with subsequent drying at reduced
pressure until constant mass.
protonated TAGs.
resulted in pronounced reduction of protonated lipids
in the prevention of promptly generated lipid fragments.
Gidden et al. speculated that base addition scavenges most of
By contrast, NaCl addition neither
[10]
nor
[13]
+
Spectrophotometry
the free
H
ions donated by the used matrix 2,5-
dihydroxybenzoic acid (DHB) thereby reducing the amount of
initial analyte protonation. However, it was not detailed if
proton scavenging should occur in solution or in the gas phase.
Obviously, the addition of a strong base during aqueous sample
preparation generates the corresponding matrix salt which
upon laser excitation should lead to generation of the cationized
matrix species instead of the protonated one thereby preventing
initial TAG protonation and fragmentation. Consequently, we
directly applied the (near) neutral matrix salts sodium
Solution absorption spectra were recorded using a dual
beam spectrophotometer (UV-2102PC, Shimadzu, Duisburg,
Germany). DHB and both DHB salts were dissolved to
À4
concentrations of 2.08 × 10 M in MeOH which was also used
as reference. Solid-phase absorption spectra were measured in
diffuse reflection mode using an integrating sphere (ISR-260,
Shimadzu). In total, 200 mg of matrix powder was mixed with
1
(
g of BaSO
Perkin-Elmer, Waltham, MA, USA) and pressed to a pellet. A
neat BaSO pellet served as the reference.
4
each finely ground in a mortar and a ball mill
2,5-dihydroxybenzoate (NaDHB) and ammonium 2,5-
4
dihydroxybenzoate (NH DHB) for sample preparation to
4
avoid extensive analyte (ester) hydrolysis due to strongly basic
sample conditions as well as to simplify the sample preparation
and for a more detailed analysis of the underlying mechanisms.
The properties of the two ionic matrices were characterized and
analyzed in comparison to standard DHB and basic DHB +
HPTLC separation
HPTLC silica gel 60 plates were purchased from Merck
(
(
Darmstadt, Germany). Heptane/diethyl ether/formic acid
80:22:2, v/v/v) was used as the solvent system to separate
free cholesterol (as a potential impurity) and cholesteryl
linoleate. The cholesterol solution (2 μL) and the cholesteryl
linoleate solution (3 μL) (see below for sample preparation)
were applied onto the silica plate. Lipids were monitored
after chromatography by spraying the TLC plate with the
NaOH/NH preparations by means of a multitude of different
3
lipid classes such as sterols, steryl esters, monoacylglycerols,
TAGs and several PCs and PEs which are abundant in
biological samples and result in characteristic differences in
their mass spectra. Therefore, this study exceeds the data
[16]
[14,15]
dye primuline as essentially described in White et al.
discussed in previous publications
by far.
Preparation of hen egg yolk extract
EXPERIMENTAL
This extract was prepared and characterized as recently
[17]
described.
In brief, hen eggs were purchased from a local
Chemicals
supermarket and twelve different eggs were extracted and
the individual extracts pooled.
All chemicals were obtained in the highest available
purity from Sigma-Aldrich (Taufkirchen, Germany) except 1-
palmitoyl-2-oleoyl-sn-glycero-3-phosphoethanolamine (16:0/18:1,
POPE) which was purchased from AVANTI Polar Lipids
The egg yolk was extracted according to the Bligh and Dyer
method, i.e., a ratio between CHCl , CH OH and the aqueous
3
3
[18]
egg yolk of 1:1:1 (v/v/v) was used. The total amount of all
extracted lipids (ca. 20 mg/mL) was determined by
weighing, whereas the phospholipid content (ca. 7 mg/mL)
(Alabaster, AL, USA). MilliQ water was prepared using a water
purification system from Millipore (Schwalbach, Germany).
31
was determined by
spectroscopy.
P nuclear magnetic resonance
[17]
Preparation of DHB salts
DHB sodium salt – sodium 2,5-dihydroxybenzoate
A slightly modified preparation procedure according to Cvacka
Sample preparation
4
DHB, NaDHB, and NH DHB were dissolved in MeOH to
[15]
and Svatos
was used. DHB (1 g; MW= 154.12 g/mol,
concentrations of 80 mM. All pure lipids were dissolved in
CHCl /MeOH (3:1, v/v) to 2.5 mg/mL by sonication for
6.49 mmol) was dissolved in MeOH (5 mL), treated with
3
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Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363

Influence of DHB-salts on lipid ionization
min; the crude hen egg yolk extract was diluted to 10 mg/mL.
Polished stainless steel MALDI targets were used for all
experiments. For analysis of the pure matrix compounds,
5
the bathochromic shift of the solid-state absorption profiles is
probably caused by intermolecular π–π orbital overlapping
which enables absorption of lower energy photons and allows
for sufficient absorption also at 355 nm. Consequently, the
extinction coefficients for the absorption measurements in
solution at the standard irradiation wavelengths of λ = 337 nm
0
.5 μL of the respective matrix solution was spotted onto the
target and air-dried. To prevent analyte spreading, 1 μL of
water was spotted onto the target followed by 0.5 μL analyte
and 0.5 μL matrix solution and air-dried. In cases of base or
salt addition, 1 μL of a 100 mM or 1 M aqueous NaOH/NH3
or NaCl solution was spotted instead of pure water which
resulted in an analyte/base/salt matrix ratio of 1:2:1 (v/v/v).
À1
À1
À1
À1
decrease from 3900 L mol cm (DHB) to 3200 L mol cm
À1
À1
(NaDHB) and 3000 L mol cm (NH DHB) and at λ = 355 nm
4
À1
À1
À1
À1
from 2300 L mol cm (DHB) to 600 L mol cm (NaDHB
and NH DHB). Irrespective of the influence of the crystal
4
morphology, a reduced absorption requires higher laser pulse
[22]
energies for highest analyte ion yields which led to a pulse
Mass spectrometry
energy increase of about 0.3 μJ/pulse for NH DHB and
4
All MALDI MS measurements were carried out using a
standard Voyager DE-STR TOF mass spectrometer (Applied
Biosystems, Darmstadt, Germany) with a 337 nm nitrogen
laser (LSI Laser Science, Newton, MA, USA) producing 3 ns
laser pulses with a pulse repetition rate of 20 Hz. The spot
size was approximately circular with 50–100 μm in diameter.
For all experiments, 500 single mass spectra were
accumulated. Pulse energies were optimized for highest
analyte S/N ratios and were 1.70 ± 0.13 μJ/pulse for DHB,
0.6 μJ/pulse for NaDHB compared to the 1.7 μJ/pulse detected
as optimum value for DHB free acid at 337 nm.
SI Figure 3(a) (Supporting Information) shows a typical
DHB mass spectrum which is in excellent agreement with
[
23]
DHB matrix spectra known from the literature.
The
•
+
molecular ion DHB
and protonated molecular species
+
[DHB + H] are detectable with significant abundances as
well as the dehydrated protonated monomer (the base peak)
at m/z 137.11 and dimer at m/z 273.04. Contrary to this, no
dehydrated products at m/z 137.11 and 273.04 (all given m/z
values refer to the monoisotopic species) are detectable in
the case of NaDHB (SI Fig. 3(b), Supporting Information).
The energetically most favorable protonation site of DHB free
1
4
.98 ± 0.13 μJ/pulse for NH DHB, and 2.29 ± 0.13 μJ/pulse
for NaDHB. For analysis of the pure DHB derivatives, pulse
energies were lowered by about 0.25 μJ/pulse for all three
compounds. A calibrated energy probe (Nova laser power/
energy meter; Ophir Optronics, Grosshansdorf, Germany)
with a low-energy pyroelectric photodiode PE10 inserted in
the laser beam path before entrance into the vacuum of the
mass spectrometer was used for determination of absolute
pulse energies (see SI Fig. 1, Supporting Information). The
chosen MALDI-TOF parameters were polarity, positive;
operation mode, reflector; accelerating voltage, 20 kV; grid
voltage, 70%; low mass gate, off; delay time, 150 ns; shots
per spectrum, 500; bin size, 0.5 ns. Data Explorer 4.5C
[24]
acid is at its carboxylic acid group.
An intramolecular
proton shift within the protonated carboxylic acid group
+
generates the dehydration products [nDHB + H–H
oxonium ion structure.
2
O] with
[23]
This rearrangement reaction is not
possible when NaDHB is used since the dominating species
+
[DHB–nH + (n + 1)Na] at m/z 177.02, 199.00, 220.98, and
+
242.96 exhibit Na chelation between the acid carbonyl
[25]
oxygen and the 2-hydroxy oxygen
and, therefore, cannot
induce loss of water. In addition to the sodiated DHB species,
+
(Applied Biosystems, Darmstadt, Germany) was used for
an intense free Na peak can be detected as well as several
spectra analysis. Peak detection was performed by using a
S/N threshold of 10 after external and internal mass
calibration and advanced baseline correction (peak width, 6;
flexibility, 1; degree, 1). Although we are aware that some
lipids would be more accurately detectable by negative ion
MALDI MS, we will focus here exclusively on positive ion
spectra because DHB and its derivatives are not the matrices
sodiated DHB fragments with low intensity which can also
be detected in the negative ion mode (SI Fig. 4, Supporting
Information) and suggests a destabilization of the molecular
+
DHB structure upon Na chelation.
•
+
The molecular ion DHB and protonated DHB are only of
low intensity in the pure NaDHB spectrum and might be due
to partial neutralization of dissolved DHB anions during
sample preparation. Pure NaDHB does not generate matrix
peaks above m/z 250 which facilitates low molecular weight
[19]
of choice to detect negative ions.
analyte peak detection. An analysis of the NH DHB spectrum
4
(
SI Fig. 3(c), Supporting Information) uncovers two
RESULTS AND DISCUSSION
+
interesting facts: first, although [DHB + NH4] generates a
strong signal, the base peak refers to sodiated DHB. Second,
Analysis of pure matrix compounds
NH DHB generates intense sodiated clusters of the structure
4
+
À
The UV-absorption properties of the generated DHB salts
were determined and compared to DHB as free acid. As
was recently shown for matrices of the α-cyanocinnamic acid
type, deprotonation going along with carboxylate formation
induces a hypsochromic shift of about 15–25 nm. . In
agreement with this, both DHB-salt absorption profiles are
hypsochromically shifted compared to that of the free acid
by about 10 nm in the solid state as well as in solution, see
SI Fig. 2 (Supporting Information). As is known from the
literature, the solid state profiles are broadened due to an
[nDHB–nH + (n + 1)Na] and [nDHB–nH + (n-1)Na] up to
heptamers (mass range not shown) in the positive and
negative ion mode (SI Fig. 4, Supporting Information) which
+
seem to be stabilized by bridging Na . This leads to the
[
20]
questions why similar clusters cannot be detected using
NaDHB and why the NH
sodiated matrix peaks. The ammonium bicarbonate used for
NH DHB generation exhibited a purity ≥99.5% with traces
4
derivative generates intense
4
of sodium ions below 0.005% and can be excluded as source
of Na contamination. All used chemicals were of the highest
purity available. No efforts were made to further reduce the
sodium contents of the used solvents and compounds.
[20,21]
enhanced degree of scattering in the solid state.
Compared to the absorption profiles recorded in solution,
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363
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T. W. Jaskolla, K. Onischke and J. Schiller
However, it cannot be excluded that residual sodium (as a
ubiquitous element) contributes to some extent to the
observed effects. Contrary to sodium salts, ammonium salts
can neutralize into the free acid form and the amine via
(cholesteryl linoleate) exhibits a peak at m/z 671.57 which refers
to the corresponding sodium adduct (Fig. 1, bottom left). It is
reasonable to assume that the cholesteryl linoleate ester
function exhibits a higher sodium cation binding affinity than
does the hydroxyl group of either cholesterol or cholestanol
resulting in the generation of the sodiated cholesteryl ester.
However, no protonated species can be detected under these
conditions. The mass spectrum of the ester exhibits an
additional peak at m/z 369.35 which was also detectable in the
mass spectrum of pure cholesterol and refers to the same
fragment, presumably resulting from cleavage of neutral
linoleic acid after initial protonation of the cholesteryl linoleate
carbonyl oxygen. To exclude that a cholesterol contamination in
the analyzed cholesteryl linoleate sample is responsible for this
peak, the used cholesteryl ester was additionally analyzed
by TLC. SI Fig. 5 (Supporting Information) shows an image
of a TLC plate subsequent to primuline staining with
[26]
proton transfer during the desorption process.
The
corresponding dissociation of NH DHB to DHB and
4
ammonia seems to be efficient since only one moderately
+
intense [DHB + NH ] peak can be detected whereas NaDHB
4
+
generates a multitude of different [DHB + Na] species.
Although the energetic conditions of this reaction are
unknown, it is reasonable to assume that NH3 release is
endothermic due to the required elimination of numerous
hydrogen bonds between NH and DHB, thereby stabilizing
3
(cooling) the matrix (cluster). Such elimination processes
could explain the detected intense matrix clusters and would
+
also stabilize Na adducts as a possible explanation for their
significant intensities.
pure cholesterol applied at lane (a) (R
pure cholesteryl linoleate at lane (b) (R
F
~0.13) and the putative
~0.92). Since no free
F
Analysis of sterol lipids
cholesterol is detectable in lane (b) we conclude that the peak
detected at m/z 369.35 in Fig. 1, bottom left is indeed due to a
gas-phase cleavage of cholesteryl linoleate and not caused by
free cholesterol impurities in the cholesteryl ester sample. The
peak at m/z 437.19 presumably refers to a cholesteryl linoleate
fragment since it can be detected with all matrices but it was
not possible to assign this mass to a certain fragment structure.
The mass spectrum of pure cholesterol recorded with DHB
exhibits a sole analyte peak at m/z 369.35 referring to
the protonated and dehydrated species [cholesterol + H–H
+
[27]
2
O]
(
see Fig. 1, top left). Neither protonated (expected at
m/z 387.36) nor sodiated (expected at m/z 409.34) cholesterol
species are detectable, the nearby peak at m/z 409.06 refers to
+
+
[
3DHB + H–3H
2
O] . The same holds true for 5α-cholestan-3β-
Whereas DHB as matrix generates strong signals of [DHB + H]
ol (cholestanol), i.e. the dihydrocholesterol derivative, with a
ions which seemingly protonate the cholesterol and
cholestanol hydroxyl group followed by instantaneous
dehydratization, such fragmentations are not observable at
all when NaDHB is used as matrix (see Fig. 1, top and middle
sole analyte peak at m/z 371.37 referring to [cholestanol+ H–
+
H
2
O] (see Fig. 1, middle left). Contrary to these lipids, the
DHB mass spectrum of the linoleic acid cholesterol ester
Figure 1. Positive ion mode MALDI-TOF mass spectra of cholesterol, cholestanol, and cholesteryl linoleate
from top to bottom) recorded in the presence of DHB (left) and NaDHB (right).
(
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Influence of DHB-salts on lipid ionization
right). Compared to DHB, NaDHB generates only very small
amounts of [DHB + H] which strongly supports the
(see Fig. 2 and SI Fig. 8, Supporting Information for
1-palmitoyl-2-oleoyl-sn-phosphatidylcholine (POPC, PC
+
hypothesis that initial analyte protonation causes the
observed fragmentation. In accordance with this, proton-
induced fragmentation of cholesteryl linoleate to [cholesteryl
4
16:0/18:1)). In the order from DHB over NH DHB to NaDHB,
+
the intensity of [PC + Na] increases strongly relative to that
+
of [PC + H] which gets continuously weaker and (nearly)
+
linoleate + H–linoleic acid] at m/z 369.35 is also strongly
cannot be detected anymore when NaDHB is used. These
+
diminished (see Fig. 1, bottom right). The low residual
intensity of this fragment is probably due to a higher
susceptibility of the cholesteryl linoleate ester function to
protonation compared to the cholesterol/cholestanol
hydroxyl group. Due to the high sodiation efficiency of
NaDHB the cleaved linoleic acid can be detected with low
intensity trends reflect the initially generated [matrix + H]
amounts of the respective DHB derivatives and allow for
simplified spectra interpretation (even without additional
tandem mass spectrometry (MS/MS)) in the case of lipid
species normally detectable as protonated as well as
cationized species in combination with NaDHB.
+
intensities as [linoleic acid + Na] at m/z 303.23 and [linoleic
Cleavage of one fatty acyl residue as neutral fatty acid is
+
+
[12,32]
acid–H + 2Na] at m/z 325.21 whereas [linoleic acid + H] at
m/z 281.25 can be detected in the case of DHB (SI Fig. 6,
Supporting Information). As is also observed in the case of
DHB, sodiated cholesteryl linoleate generates a strong signal
at m/z 671.57 using NaDHB. However, sodiated cholesterol/
commonly observed in MS/MS mode.
However, the
+
corresponding fragments [PC–fatty acid] can also be
detected with at least low intensities for all investigated PCs
with the three DHB compounds, see m/z 478.33 and 504.34
in Fig. 3 (POPC) and m/z 478.33 in SI Fig. 9 (Supporting
Information, dipalmitoyl-sn-phosphatidylcholine, DPPC, PC
16:0/16:0, data not shown for DSPC). With DHB as
4
cholestanol is still not detectable. If NH DHB is used, the
spectra resemble mixtures between that of DHB and NaDHB
+
+
resulting in low intensities of [cholesterol+ H–H
2
O] (see SI
protonating matrix, the fragment [PC–fatty acid] is generated
Fig. 7, Supporting Information). This is most probably due to
by cleavage of a free fatty acid from the protonated lipid. The
correct annotation is, therefore, [PC + H–free fatty acid] . By
+
+
the ammonium ion of [DHB + NH
4
]
which, in addition to
+
[28–31]
[DHB + H] , may also lead to analyte protonation
and
contrast, in the case of NaDHB with (nearly) exclusive
generation of the intact sodiated lipid, the same fragment
once again supports the idea of initial analyte protonation as
the source for the detected prompt fragmentation.
+
must correspond to [PC + Na–fatty acid sodium salt] .
Regarding the underlying pathway, it was suggested that
an intramolecular proton shift from the glycerol backbone
to one of the fatty acyl carbonyl oxygen atoms leads to
Analysis of phosphatidylcholines
+
+
Suppression of initial analyte protonation can also be
detected in the case of phosphatidylcholines (PCs). As a
consequence, PC fragmentations induced by protonation are
also prevented. Distearoyl-sn-phosphatidylcholine (DSPC,
PC 18:0/18:0) can be detected as intense protonated species
in addition to low amounts of sodiated DSPC using DHB
[PC + Na–free fatty acid] with the Na ion still attached
[
33,34]
at the PC residue.
Since these fragment ions exhibit
only extremely low intensities (about 1% in comparison
to the molecular PC peaks) or are not detectable at all, this
pathway seems to be only of minor relevance under
MALDI-MS conditions; see m/z 500.31 and 526.32 in Fig. 3
Figure 2. Positive ion mode MALDI-TOF mass spectra of pure DSPC recorded
in the presence of DHB (a), NH DHB (b), and NaDHB (c). The chemical
4
structure of the zwitterionic form of DSPC is shown in the bottom trace.
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T. W. Jaskolla, K. Onischke and J. Schiller
Figure 3. Positive ion mode MALDI-TOF mass spectra of pure POPC recorded
with DHB (a) and NaDHB (b) as matrices. Analyte m/z ratios are given in bold.
and SI Fig. 8 (Supporting Information) with complete absence
no detectable peaks at m/z 206.06 (referring to NaHPO
4
+
+
of the [DPPC + Na–free palmitic acid] species at m/z 526.32.
Al-Saad et al. proposed an alternative pathway in which the
negatively charged phosphate oxygen intramolecularly attacks
one of the two carbon atoms connected to the fatty acyl
(CH
DHB or NH
2
)
2
N(Me)
3
) if NaDHB is used compared to corresponding
DHB experiments with clearly more intense
4
fragment formation at m/z 184.07 (see SI Fig. 10, Supporting
Information) for DSPC as analyte.
Fragmentations induced by intramolecular proton shifts
which are not suppressed by sodium cation binding yield
nearly identical fragment intensities for DHB, NH DHB,
4
and NaDHB. This becomes obvious for the cleavage of the
[
35]
residues.
With a more electrostatically than covalently
bound sodium ion, this pathway leads to loss of fatty acids as
neutral sodium salts and positively charged cyclic ions in
accordance with the observed fragments. The negatively
charged phosphate oxygen of sodiated PCs facilitates fatty acid
cleavage and explains the higher intensities of fragmentation
permanently charged trimethylvinylammonium cation
+
3
H
2
C = CH-N(CH
3
)
at m/z 86.10 generated by an intramo-
products when NaDHB or NH
DHB which generates predominantly protonated PCs with
covalently bound phosphate O-H groups.
Gas-phase elimination of PL fatty acids as ketenes as possible
explanation for the peaks at m/z 496.34 and 522.35 requires
collisionally activated dissociation (CAD) conditions.
can be expected that these peaks refer to small amounts of
4
DHB is used compared to
lecular proton shift from the acidified methylene group
connected to the trimethylammonium head group to one of
the negatively charged phosphate oxygens (SI Scheme 1 and
SI Fig. 10, Supporting Information). The rearrangement is
spatially unhindered also in the case of bound sodium
cations. The same applies for generation of [PC–polar head]
ions with diacylglycerol-like structures for which one of the
negatively charged phosphate oxygens is formally attacked
[36,37]
+
It
[38]
lysophosphatidylcholines (LPCs) contaminating the analyte,
i.e., [lyso-1-palmitoyl-sn-phosphatidylcholine + H] at m/z
+
by a methylene cation generated upon loss of trimethylamine.
+
+
496.34 and [lyso-2-oleoyl-sn-phosphatidylcholine + H] at m/z
The intermediate species [PL–N(Me) ] can be detected in the
3
5
22.35 (see Fig. 3) as well as [lyso-1/2-palmitoyl-
MS spectra, a subsequent rearrangement leads to formation
of diacylglycerol-like cations which are stabilized by
+
sn-phosphatidylcholine+ H] at m/z 496.34 (see SI Fig. 9,
Supporting Information). The corresponding sodiated species
[35]
cyclization (SI Scheme 2, Supporting Information).
Again,
+
[lyso-1-palmitoylphosphatidylcholine + Na] at m/z 518.32 and
these rearrangements are sterically unhindered by sodium
cationization and result in comparable absolute fragment
intensities for all three DHB derivatives, see Fig. 3 for
+
[lyso-2-oleoyl-sn-phosphatidylcholine+ Na] at m/z 544.34 are
detectable with extremely low intensities in the spectra of POPC
recorded in the presence of NaDHB.
Transfer of a proton from the glycerol backbone to the
phosphate oxygen of protonated PCs leads to a cyclic
+
[POPC–polar head] at m/z 577.52 and SI Fig. 9 (Supporting
+
Information) for [DPPC–polar head] at m/z 551.50.
These examples clearly indicate that NaDHB (and, to a
rearrangement with elimination of the charged phosphate
4
limited extent, NH DHB) not only suppresses initial analyte
+
head group H
2
PO
4
2
(CH )
2
N(Me)
3
at m/z 184.07. As was
protonation, but also inhibits analyte fragmentation if the
+
observed previously, such rearrangements are strongly
restricted in the case of sodiated PCs in which the phosphate
respective rearrangement is sterically hindered by bound Na .
[35,39]
group is stabilized by a sodium cation.
This can be
Analysis of phosphatidylethanolamines
explained by steric hindrance in combination with a lower
charge density of the proton-uptaking phosphate oxygen
due to sodium complexation and the required energy for
breaking the electrostatic interaction between the sodium
cation and the carbonyl-oxygens. In agreement with these
results, analyses of DPPC, DSPC, and POPC yielded almost
Phosphatidylethanolamines are typically detectable as
protonated as well as singly and doubly sodiated species, see
Fig. 4(a) for an exemplary mass spectrum of 1-palmitoyl-2-
oleoyl-sn-phosphatidylethanolamine (POPE) recorded with
DHB. This multitude of different ion states for one single
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363

Influence of DHB-salts on lipid ionization
Figure 4. Positive ion mode MALDI-TOF mass spectra of pure POPE recorded
with DHB (a) and NaDHB (b).
compound reduces the achievable sensitivity and can be
avoided by using NaDHB which almost exclusively generates
the doubly sodiated species [PE–H + 2Na] , see Fig. 4(b) for
POPE and SI Fig. 11 (Supporting Information) for 1,2-
distearoyl-sn-phosphatidylethanolamine (DSPE) as analyte.
Analysis of acylglycerols
+
Glyceryl trioleate (triolein) was investigated as representative
of TAGs. Although we will not investigate complex lipid
mixtures here, all the discussed lipids were chosen because
they are very abundant in biological systems such as human
4
As was the case for PCs, NH DHB behaves like a mixture
[40]
lipoproteins.
As common for TAGs, triolein cannot be
between DHB and NaDHB which results in intense sodiated
PE species with additional weaker signals of the protonated
analytes.
protonated under MALDI-MS conditions and is only
detectable as sodiated species for all three DHB derivatives
(
see SI Fig. 12, Supporting Information). In agreement with
As was shown before for the lipid classes of sterols and
PCs, prevention of analyte protonation using NaDHB can
suppress fragmentation processes. This is also true for PEs
previously discussed sterol fragmentation behavior, DHB
induces prompt TAG fragmentation with generation of
DAG-like [triolein + H–oleic acid] ions at m/z 603.53. With
+
+
with inhibition of [PE–polar head] generation, see Fig. 4
+
NH DHB, weak [triolein + H–oleic acid] intensities are also
and SI Fig. 11 (Supporting Information). The corresponding
4
+
detectable whereas no fragmentation at all is observable in
the case of NaDHB. As outlined before, this is a compelling
piece of evidence that these fragmentations are induced by
species [PC–polar head] were detectable by all three DHB
derivatives due to their permanently charged PC choline
head groups which can be eliminated as neutral
trimethylamine (see SI Scheme 2, Supporting Information).
Only in the case of protonated PEs a corresponding+
elimination of ammonia as initial step of [PE–polar head]
generation is possible; sodiated precursors are insusceptible to
such rearrangements. For the same reason, cleavage of
+
initial analyte protonation either by [DHB + H] in the case
+
of DHB or [DHB + NH ] in the case of NH DHB. The
4
4
+
possibility that NH4 ions induce analyte protonation with
subsequent fragmentation has already been observed for
[
31]
TAG ammonium adducts,
which led to DAG-like ions
+
3
identical to the ones detected in the experiments discussed
in this paper. These results are in agreement with Gidden
et al. who demonstrated that DAG-like fragments can be
suppressed by addition of highly concentrated NaOH or
protonated vinylamine H C = CH-NH which typically
2
generates an intense peak at m/z 44.05 in combination with
DHB is fully suppressed if NaDHB (or restricted in the case of
NH DHB) is used. Due to the lack of negative charge density,
4
NH
3
solutions to DHB preparations which led to in situ
also sodiated vinylamine cannot be detected. One could expect
that neutral (and therefore not detectable) vinylamine still gets
[
13]
NaDHB or NH
4
DHB matrix-salt generations.
In addition,
+
matrix-free analysis of cuticular TAG lipids under UV-LDI
conditions resulted in exclusive detection of the corresponding
intact potassium adducts without any DAG-like fragments in
eliminated. In this case, [PE–H + 2Na–vinylamine] should be
detectable in the NaDHB spectra of PEs. However, the intensity
of these fragments is extremely low, if detectable at all, which
suggests that neutral vinylamine elimination is also strongly
restricted. It can be assumed that the intramolecular proton
rearrangement as shown in the example of PCs in SI Scheme 1
[
41]
accordance with the results presented in this work.
Since no peak was detectable at m/z 603.53 using triolein as
analyte and NaDHB as matrix, elimination of sodium oleate
from the sodiated precursor leading to [TAG + Na–fatty acid
(see Supporting Information) is impeded when both free
+
sodium salt] does not occur. This is in contrast to PCs, which
phosphate oxygens are electrostatically bound to the two
+
+
generate [PC + Na–fatty acid sodium salt] if analyzed with
[
PE–H + 2Na] sodium cations which would lead to steric
NaDHB (Fig. 3). This different behavior can be explained by
the negatively charged phosphate oxygens of sodiated PCs
which exhibit higher nucleophilicities than the neutral
carbonyl oxygens of TAGs and, therefore, can undergo a
hindrance and lowered negative phosphate oxygen charge
densities. These fragmentation suppressions and spectra
simplifications are further examples for the effects of analyte
sodiation using NaDHB.
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363
Copyright © 2014 John Wiley & Sons, Ltd.
wileyonlinelibrary.com/journal/rcm

T. W. Jaskolla, K. Onischke and J. Schiller
more efficient intramolecular rearrangement. This implies
well as their fragments, if detectable. The selected mass range
shows a multitude of signals referring to protonated PCs and
PEs in addition to several sodiated TAGs. A detailed analysis
of the detectable lipid species is given in SI Table 1 (see
Supporting Information). As was demonstrated by means of
several pure PC and PE species as well as isolated triolein,
NaDHB enables nearly exclusive detection of sodiated lipids
and exhibits a high sodiation efficiency leading to detection
of, e.g., oleoylglycerol. This also applies for the investigated
lipid mix from egg yolk, the protonated PCs detected with
DHB at m/z 758.6 and 760.6 are (nearly) not detectable
using NaDHB. By contrast, a wealth of sodiated PCs and
PEs can be detected in the range from m/z 762 to 832. Most
of the peaks in this mass range can be attributed to
protonated as well as sodiated PC/PE-species which impedes
unambiguous peak correlation in the case of DHB (cf. SI
Table 1, Supporting Information). As previously shown,
NaDHB allows for exclusive detection of sodiated species.
The only exception might be the weak signal at m/z 760.53
in the NaDHB spectrum which can be attributed to
protonated PC 16:0/18:1 and/or 16:1/18:0 (m/z 760.59, cf.
the corresponding intense peak in the DHB spectrum) but
+
that fragmentation of [triolein + Na] to [triolein + Na–sodium
+
oleate] as an alternative explanation for the detected m/z
6
03.53 using DHB can be excluded.
-Oleoyl-rac-glycerol (OG) as an example for monoacy-
1
lglycerols (MAGs, which may be generated from TAGs under
physiological conditions upon lipase digestion) was also
investigated by means of the three DHB derivatives (see
Fig. 5). The peak at m/z 361.88 in the DHB spectrum can also
be detected in pure DHB matrix spectra and seems to refer to
an unassigned matrix peak. As was the case for TAGs, OG
protonation cannot be observed due to lack of basicity. Since
MAGs exhibit only one fatty acyl residue and, therefore, only
one ester function with high negative charge density, their
cationization is more difficult than that of TAGs. As a result,
the intact sodiated species can only be detected using the
4
stronger cationizing agent NH DHB or NaDHB at m/z 379.28
whereas this species was not detectable using DHB. Interestingly,
NaDHB with the highest sodium cation transfer efficiency of
the investigated derivatives even generates doubly sodiated
+
oleoyl glycerol [OG–H + 2Na] at m/z 401.26 with low intensity.
Due to its energetically unfavorable alcoholate structure, this
+
ion can undergo fragmentation leading to generation of
might be also caused by PE–H + 2Na 16:1/18:1 (m/z
+
[
oleic acid–H + 2Na] detectable at m/z 327.23 with low
760.49) which can be detected in its protonated form as a
low intensity peak in the DHB spectrum. The (nearly)
quantitative generation of sodiated species greatly simplifies
peak correlation, as is demonstrated by the example of m/z
intensity (see Fig. 5(c)). The DHB spectrum exhibits a nearby
peak at m/z 326.32 which does not refer to this fragment ion.
7
90.57 which can be correlated to a sodiated PE with four
Analysis of real-life lipid samples
double bonds (PE 18:0/20:4) at m/z 790.54 or to a saturated
protonated PC at m/z 790.63 (for more details, see SI Table 1,
Supporting Information). Since this peak can only be detected
with NaDHB, it has to refer to the sodiated PE species. In
addition, its protonated counterpart at m/z 768.55 can be
detected with low intensities using DHB. Furthermore, if
m/z 790.57 would refer to a protonated PC, its sodiated
To verify the discussed beneficial properties of DHB salts on
the example of a "real life" sample, a crude organic hen egg
yolk extract was investigated using DHB and NaDHB as
matrices (see Fig. 6). Both mass spectra were recorded in the
positive ion mode and internally calibrated by means of
protonated and sodiated POPC, POPE, DPPC, and DSPC as
Figure 5. Positive ion mode MALDI-TOF mass spectra of pure OG
recorded with DHB (a), NH DHB (b), and NaDHB (c). The region around
4
m/z 327.23 is emphasized in (c).
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Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363

Influence of DHB-salts on lipid ionization
Figure 6. Positive ion mode MALDI-TOF mass spectra of a crude egg yolk extract recorded
with DHB (a) and NaDHB (b).
counterpart at m/z 812.61 should exhibit significantly
stronger intensities if NaDHB is used as matrix, which is,
however, not the case.
not observable at all if the (near) neutral matrix salts NaDHB/
NH DHB are used (pH of 1 M aqueous solutions: NaDHB, ≈7;
NH DHB, ≈5.5). NaOH addition exceeding stoichiometric
4
4
The high sodiation efficiency of NaDHB also allows for
simplified TAG detection, as can be seen in the range starting
from about m/z 850. In comparison to DHB, numerous and
more intense sodiated TAGs with one to five double bonds
are detectable using NaDHB. In a nutshell, more detailed
information about the lipid composition of a complex sample
can be obtained when the most appropriate matrix is used.
matrix amounts leads to free excess NaOH which impedes
matrix crystallization and further lowers the achievable analyte
intensities. Addition of insufficient base amounts, however, does
not fully suppress fragmentation resulting from initial analyte
protonation. The optimum base amount for suppression of
complete prompt fragmentation with simultaneously minimized
analyte hydrolysis was determined at about 100 mM NaOH. If
base should be added to DHB preparations, it is recommended
to use NaOH rather than NH , which after conversion into
3
Comparison between NH DHB/NaDHB and NH /NaOH or NaCl
addition to DHB sample preparations
4
3
NH DHB can cause analyte protonation and fragmentation
4
although the efficiency is lower than that of DHB. In addition,
sample preparations should be done as quickly as possible to
reduce analyte hydrolysis under alkaline conditions.
Gidden et al. demonstrated that TAG fragmentation can be
[13]
eliminated by base addition to the matrix/sample mixture
which generated the corresponding matrix salts during
sample preparation. It was reported that the added bases
did not impede formation of sodiated TAGs. However, upon
addition of bases at concentrations recommended for
complete suppression of proton-induced prompt analyte
As expected, addition of NaCl results in increased lipid
sodiation, see SI Fig. 14 (Supporting Information) for the
example of egg yolk extract analyzed with DHB in the
presence of 100 mM or 1 M NaCl. However, also upon
addition of 1 M NaCl it was not possible to significantly
reduce the protonated lipid intensities or their fragmentation
products, respectively. Higher salt concentrations could not
be used due to insufficient spectra quality. Therefore, salt
addition might be useful for analysis of lipids which can only
be cationized but do not suppress proton-induced lipid
fragmentation or allow for simplified lipid identification.
[13]
fragmentations (1 M NaOH or 14 M NH
3
), pronounced
alkaline analyte hydrolysis was observed. If sample drying
exceeded a few minutes even total analyte degradation was
observable (see SI Fig. 13, Supporting Information) for OG
as analyte prepared with DHB and 1 M NaOH using a drying
time of 10 min. In addition to low or missing intact analyte
ion intensities, analyte hydrolysis can also be recognized by
intense fragment peaks of the sodiated hydrolyzed fatty
+
acids, e.g., [oleic acid–H + 2Na] in the case of OG as analyte
CONCLUSIONS
(SI Fig. 13, Supporting Information). The corresponding analyte
counterpart is also observable as cationized species, e.g., sodiated
glyceryl dioleate (diolein) and [OG + Na] in the case of triolein.
The differences in ionization behavior between classic DHB
and the DHB salts NH DHB and NaDHB were investigated
4
+
These fragments exhibit only extremely low intensities or are
by means of a multitude of lipid classes. Suppression of
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363
Copyright © 2014 John Wiley & Sons, Ltd. wileyonlinelibrary.com/journal/rcm

T. W. Jaskolla, K. Onischke and J. Schiller
several analyte fragmentations were detected if the DHB salts
were used. Two types of suppressed fragmentations have been
identified. First, fragmentations induced by (initial) analyte
protonation as was detected for, e.g., sterols which otherwise
dehydrate, TAGs which undergo fatty acid cleavage or PEs with
loss of the polar head group, and, second, fragmentations
suppressed by steric hindrance of the bound sodium cation(s).
[3] K. Simons, R. Ehehalt. Cholesterol, lipid rafts, and disease.
J. Clin. Invest. 2002, 110, 597.
[
4] G. J. Siegel, B. W. Agranoff, R. A. Albers, S. K. Fisher,
M. D. Uhler. Basic Neurochemistry, Molecular, Cellular and
Medical Aspects, Lippincott-Raven, Philadelphia, 1999.
5] M. Pulfer, R. C. Murphy. Electrospray mass spectrometry of
phospholipids. Mass Spectrom. Rev. 2003, 22, 332.
6] S. H. Lee, M. V. Williams, I. A. Blair. Targeted chiral lipidomics
analysis. Prostaglandins Other Lipid Mediat. 2005, 77, 141.
[
[
Examples for the latter are elimination of the charged phosphate
+
3
head group NaHPO (CH ) N(Me) from PCs or PE vinylamine
4
2 2
[7] J. Schiller, R. Süß, B. Fuchs, M. Müller, O. Zschörnig,
K. Arnold. MALDI-TOF MS in lipidomics. Front Biosci. 2007,
12, 2568.
[8] J. Schiller, J. Arnhold, S. Benard, M. Müller, S. Reichl,
K. Arnold. Lipid analysis by matrix-assisted laser desorption
elimination. In addition to restricted fragmentation patterns
and with more importance for everyday measurements,
NH DHB and especially NaDHB prevent the generation of
4
protonated molecular analyte species. This can result in
simplified mass spectra where analytes typically appear as
protonated as well as sodiated species as is the case for PCs
and especially for PEs. This was also demonstrated by using a
crude egg yolk extract as an example for a "real life" sample.
Moreover, analytes such as oleoylglycerol which neither can
be protonated nor sodiated using DHB could be detected with
high sensitivities as cationized species due to the higher sodium
cationization efficiency of the investigated matrix salts. In
comparison to DHB and on the example of an egg yolk extract,
the higher NaDHB sodiation efficiency also allowed for a more
sensitive TAG detection.
and ionization mass spectrometry:
A methodological
approach. Anal. Biochem. 1999, 267, 46.
[
9] J. Schiller, R. Süß, M. Petkovic, N. Hilbert, M. Müller,
O. Zschörnig, J. Arnhold, K. Arnold. CsCl as an auxiliary
reagent for the analysis of phosphatidylcholine mixtures
by matrix-assisted laser desorption and ionization time-of-
flight mass spectrometry (MALDI-TOF MS). Chem. Phys.
Lipids. 2001, 113, 123.
[10] R. L. Griffiths, J. Bunch. A survey of useful salt additives in
matrix-assisted laser desorption/ionization mass
spectrometry and tandem mass spectrometry of lipids:
introducing nitrates for improved analysis. Rapid Commun.
Mass Spectrom. 2012, 26, 1557.
4
NaDHB proved to be superior to NH DHB since it almost
[
11] G. R. Asbury, K. Al-Saad, W. F. Siems, R. M. Hannan,
H. H. Hill Jr. Analysis of triacylglycerols and whole oils by
matrix-assisted laser desorption/ionization time of flight
mass spectrometry. J. Am. Soc. Mass Spectrom. 1999, 10, 983.
[12] K. A. Al-Saad, V. Zabrouskov, W. F. Siems, N. R. Knowles,
R. M. Hannan, H. H. Hill Jr. Matrix-assisted laser
desorption/ionization time-of-flight mass spectrometry of
lipids: ionization and prompt fragmentation patterns. Rapid
Commun. Mass Spectrom. 2003, 17, 87.
13] J. Gidden, R. Liyanage, B. Durham, J. O. Lay Jr. Reducing
fragmentation observed in the matrix-assisted laser
desorption/ionization time-of-flight mass spectrometric
analysis of triacylglycerols in vegetable oils. Rapid Commun.
Mass Spectrom. 2007, 21, 1951.
14] R. Arakawa, N. Miyake, S. Okuno, H. Yamaoka. Signal
enhancement on laser desorption/ionization using alkali
dihydroxybenzoic acid salts as cationizing agents. Rapid
Commun. Mass Spectrom. 2006, 20, 2063.
15] J. Cvacka, A. Svatos. Matrix-assisted laser desorption/
ionization analysis of lipids and high molecular weight
hydrocarbons with lithium 2,5-dihydroxybenzoate matrix.
Rapid Commun. Mass Spectrom. 2003, 17, 2203.
[16] T. White, S. Bursten, D. Federighi, R. A. Lewis, E. Nudelman.
High-resolution separation and quantification of neutral lipid
and phospholipid species in mammalian cells and sera by
multi-one-dimensional thin-layer chromatography. Anal.
Biochem. 1998, 258, 109.
completely suppresses analyte protonation and corres-
ponding fragmentation reactions and exclusively generates
the highest possible analyte cationization state.
The investigated DHB compounds differ in protonation
efficacy whereby DHB represents the strongest protonating
4
agent followed by NH DHB and NaDHB without any
protonation power. The protonated analyte intensities as well
as the fragment intensities of assumed proton-induced
fragmentations followed this trend without exception. This
clearly proves the previously proposed idea of instable initial
analyte protonation as explanation of the observed fragment
ions. Similar results were achievable if high amounts of bases
were added to the DHB preparations. However, partially or
even total analyte hydrolysis occurred during aqueous sample
preparation in dependence on the added base amount and
sample drying time which can completely destroy the analyte.
[
[
[
Acknowledgements
Financial support by the German Research Council (DFG
Grant JA 2127/1-1 to T.W.J. and Schi 476/12-1 and SFB
1
052/B6 to J.S.) is gratefully acknowledged. These studies
were additionally supported by the Federal Ministry of
Education and Research of the Federal Republic of Germany
[
17] B. Fuchs, J. Schiller, R. Süss, M. Schürenberg, D. Suckau. A
direct and simple method of coupling matrix-assisted laser
desorption and ionization time-of-flight mass spectrometry
("The Virtual Liver", 0315735). We thank Prof. Michael Karas
from the University of Frankfurt for the possibility to use
his Voyager DE-STR mass spectrometer.
(MALDI-TOF MS) to thin-layer chromatography (TLC) for
the analysis of phospholipids from egg yolk. Anal. Bioanal.
Chem. 2007, 389, 827.
18] E. G. Bligh, W. J. Dyer. A rapid method of total lipid
extraction and purification. Can. J. Biochem. Physiol. 1959, 37, 911.
REFERENCES
[
[
1] A. Z. Fernandis, M. R. Wenk. Membrane lipids as signaling
molecules. Curr. Opin. Lipidol. 2007, 18, 121.
2] N. Ermak, B. Lacour, F. Goirand, T. B. Drueke, S. Vicca.
Differential apoptotic pathways activated in response to Cu-
induced or HOCl-induced LDL oxidation in U937 monocytic
cell line. Biochem. Biophys. Res. Commun. 2010, 393, 783.
[19] B. Fuchs, J. Schiller. Recent developments of useful MALDI
matrices for the mass spectrometric characterization of
apolar compounds. Curr. Org. Chem. 2009, 13, 1664.
[20] R. Cramer, M. Karas, T. W. Jaskolla. Enhanced MALDI MS
sensitivity by weak base additives and glycerol sample
coating. Anal. Chem. 2014, 86, 744.
[
wileyonlinelibrary.com/journal/rcm
Copyright © 2014 John Wiley & Sons, Ltd.
Rapid Commun. Mass Spectrom. 2014, 28, 1353–1363

Influence of DHB-salts on lipid ionization
[
[
21] H.-C. Lüdemann. Dissertation, University of Münster,
Münster, Germany, 2000.
[32] S. N. Jackson, H.-Y. J. Wang, A. S. Woods. In situ structural
characterization of phosphatidylcholines in brain tissue
using MALDI-MS/MS. J. Am. Soc. Mass Spectrom. 2005, 16,
2052.
[33] F.-F. Hsu, A. Bohrer, J. Turk. Formation of lithiated adducts of
glycerophosphocholine lipids facilitates their identification by
electrospray ionization tandem mass spectrometry. J. Am. Soc.
Mass Spectrom. 1998, 9, 516.
22] J. Soltwisch, T. W. Jaskolla, F. Hillenkamp, M. Karas,
K. Dreisewerd. Ion yields in UV-MALDI mass spectrometry
as a function of excitation laser wavelength and optical and
physico-chemical properties of classical and novel halogen-
substituted MALDI matrices. Anal. Chem. 2012, 84, 6567.
23] W. E. Wallace, M. A. Arnould, R. Knochenmuss. 2,5-
Dihydroxybenzoic acid: laser desorption/ionization as a
function of elevated temperature. Int. J. Mass Spectrom.
[
[
[34] P. Domingues, F. M. L. Amado, M. G. O. Santana-Marques,
A. J. Ferrer-Correia. Constant neutral loss scanning for the
2
005, 242, 13.
characterization
of
glycerol
phosphatidylcholine
24] S. Bourcier, Y. Hoppilliard. B3LYP DFT molecular orbital
approach, an efficient method to evaluate the
thermochemical properties of MALDI matrices. Int. J. Mass
Spectrom. 2002, 217, 231.
phospholipids. J. Am. Soc. Mass Spectrom. 1998, 9, 1189.
[35] K. A. Al-Saad, W. F. Siems, H. H. Hill, V. Zabrouskov,
N. R. Knowles. Structural analysis of phosphatidylcholines
by post-source decay matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry. J. Am. Soc. Mass
Spectrom. 2003, 14, 373.
[25] G. Ohanessian. Interaction of MALDI matrix molecules with
+
Na in the gas phase. Int. J. Mass Spectrom. 2002, 219, 577.
[
26] E. Nordhoff, R. Cramer, M. Karas, F. Hillenkamp, F. Kirpekar,
K. Kristiansen, P. Roepstorff. Ion stability of nucleic acids in
infrared matrix-assisted laser desorption/ionization mass
spectrometry. Nucleic Acids Res. 1993, 21, 3347.
[36] F.-F. Hsu, J. Turk. Characterization of phosphatidylinositol,
phosphatidylinositol-4-phosphate, and phosphatidylino-
sitol-4,5-bisphosphate by electrospray ionization tandem
mass spectrometry: A mechanistic study. J. Am. Soc. Mass
Spectrom. 2000, 11, 986.
[37] M. C. Thomas, T. W. Mitchell, S. J. Blanksby. A comparison
of the gas phase acidities of phospholipid headgroups:
Experimental and computational studies. J. Am. Soc. Mass
Spectrom. 2005, 16, 926.
[38] N. J. Zuidam, H. K. M. E. Gouw, Y. Barenholz, D. J. A. Crommelin.
Physical (in)stability of liposomes upon chemical hydrolysis:
the role of lysophospholipids and fatty acids. Biochim. Biophys.
Acta 1995, 1240, 101.
[39] P. Domingues, M. R. M. Domingues, F. M. L. Amado,
A. J. Ferrer-Correia. Characterization of sodiated glycerol
phosphatidylcholine phospholipids by mass spectrometry.
Rapid Commun. Mass Spectrom. 2001, 15, 799.
[
27] J. Schiller, J. Arnhold, H. J. Glander, K. Arnold. Lipid analysis
of human spermatozoa and seminal plasma by MALDI-TOF
mass spectrometry and NMR spectroscopy – effects of
freezing and thawing. Chem. Phys. Lipids 2000, 106, 145.
28] D. Barceló, M. Honing, S. Chiron. LC-MS: A new window
for trace-level monitoring of pesticides and their
transformation products in environmental samples, in
Applications of LC-MS in Environmental Chemistry. Journal
of Chromatography Library, vol. 59 (Ed: D. Barcelo),
Elsevier Science, Amsterdam, 1996, p. 226.
[
[
29] Y. F. Zhu, N. I. Taranenko, S. L. Allman, S. A. Martin, L. Haff,
C. H. Chen. The effect of ammonium salt and matrix in the
detection of DNA by matrix-assisted laser desorption/
ionization time-of-flight mass spectrometry. Rapid Commun.
Mass Spectrom. 1996, 10, 1591.
[40] A. M. Scanu, A. A. Spector. Biochemistry and Biology of Plasma
Lipoproteins. Marcel Dekker, New York, 1986.
[
30] X. J. Yang, Y. Qu, Q. Yuan, P. Wan, Z. Du, D. Chen, C. Wong.
Effect of ammonium on liquid- and gas-phase protonation
and deprotonation in electrospray ionization mass
spectrometry. Analyst 2013, 138, 659.
[41] J. Y. Yew, J. Soltwisch, A. Pirkl, K. Dreisewerd. Direct laser
desorption ionization of endogenous and exogenous
compounds from insect cuticles: practical and methodologic
aspects. J. Am. Soc. Mass Spectrom. 2011, 22, 1273.
[
31] P. J. Kalo, V. Ollilainen, J. M. Rocha, F. X. Malcata.
Identification of molecular species of simple lipids by
normal phase liquid chromatography-positive electrospray
tandem mass spectrometry, and application of developed
methods in comprehensive analysis of low erucic acid
rapeseed oil lipids. Int. J. Mass Spectrom. 2006, 254, 106.
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