Compelling evidence for lucky survivor and gas phase protonation: The unified MALDI analyte protonation mechanism

Article abstract of DOI:10.1007/s13361-011-0093-0

This work experimentally verifies and proves the two long since postulated matrix-assisted laser desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and the gas phase protonation model. Experimental differentiation betwe

Full text of DOI:10.1007/s13361-011-0093-0

B American Society for Mass Spectrometry, 2011
J. Am. Soc. Mass Spectrom. (2011) 22:976Y988
DOI: 10.1007/s13361-011-0093-0
FOCUS: MALDI-TOF AND BIOLOGICAL MS: RESEARCH ARTICLE
Compelling Evidence for Lucky Survivor and Gas
Phase Protonation: The Unified MALDI Analyte
Protonation Mechanism
Thorsten W. Jaskolla, Michael Karas
Cluster of Excellence Macromolecular Complexes, Institute of Pharmaceutical Chemistry, Goethe University Frankfurt,
Max-von-Laue-Str. 9, 60438, Frankfurt, Germany
Abstract
This work experimentally verifies and proves the two long since postulated matrix-assisted laser
desorption/ionization (MALDI) analyte protonation pathways known as the Lucky Survivor and
the gas phase protonation model. Experimental differentiation between the predicted mecha-
nisms becomes possible by the use of deuterated matrix esters as MALDI matrices, which are
stable under typical sample preparation conditions and generate deuteronated reagent ions,
including the deuterated and deuteronated free matrix acid, only upon laser irradiation in the
MALDI process. While the generation of deuteronated analyte ions proves the gas phase
protonation model, the detection of protonated analytes by application of deuterated matrix
compounds without acidic hydrogens proves the survival of analytes precharged from solution in
accordance with the predictions from the Lucky Survivor model. The observed ratio of the two
analyte ionization processes depends on the applied experimental parameters as well as the
nature of analyte and matrix. Increasing laser fluences and lower matrix proton affinities favor
gas phase protonation, whereas more quantitative analyte protonation in solution and
intramolecular ion stabilization leads to more Lucky Survivors. The presented results allow for
a deeper understanding of the fundamental processes causing analyte ionization in MALDI and
may alleviate future efforts for increasing the analyte ion yield.
Key words: CHCA, ClCCA, Deuteration, Deuteronation, Ester, Ionization, Matrix, Plume
reactions, Proton transfer
such as, e.g., laser fluence and irradiation wavelength as well as
matrix and analyte properties led to a large variety of proposals
Introduction
lthough matrix-assisted laser desorption/ionization
(MALDI) mass spectrometry (MS) [1–4] has found
widespread use in the analysis of biological and biochemical
samples, the essential analyte ionization step is not yet fully
understood. Variations of experimentally accessible parameters
and partial aspects regarding the underlying processes [2, 5–
28]. For closer insights, the physicochemical reactions taking
place were elucidated by determination of the gas phase
basicities as well as proton and electron affinities of the
involved matrix [29–36] and analyte [37–43] neutral and ion
species and were complemented by theoretical considerations
and computational chemistry [34, 44–49]. From these inves-
tigations, two conflicting theories predominantly discussed
nowadays emerged: The Lucky Survivor theory [17, 50] and
the gas phase protonation model [5, 16, 24, 28, 51]. Both will
be briefly discussed in the following.
A
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-011-0093-0) contains supplementary material, which
is available to authorized users.
Correspondence to: Thorsten Jaskolla; e-mail: Jaskolla@pharmchem.
uni-frankfurt.de
Received: 8 November 2010
Revised: 3 January 2011
Accepted: 3 January 2011
Published online: 8 March 2011

T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
977
The Lucky Survivor Model
ions lead to proton transfer reactions and to protonated or
deprotonated analytes, respectively [16, 24, 51].
The Lucky Survivor model postulates that analytes are
incorporated in the matrix crystals with their respective
charge states preserved from solution. At typical preparation
conditions, peptides and proteins, the preferentially inves-
tigated analyte classes, are protonated and would therefore
be incorporated as positively precharged analytes together
with their counterions. Incorporation of analytes in their
respective solution charge state could be confirmed by
means of pH indicator probes [52] and adduct detection of
precharged analytes with anions of extremely strong acids
originating from solution [53, 54]. The original model
differentiates between two possibilities: In the first case,
ablated clusters generated upon laser irradiation contain
precharged analytes with their corresponding amount of
counterions and are therefore without net charge. Cluster
dissociation induces entire charge neutralization or gener-
ation of net neutral adducts in the case of stable anions
which both cannot be detected. In the second case, clusters
carry a net charge initiated by charge separation upon
disruption of the matrix-analyte solid. A lack of counterions
inhibits quantitative charge neutralization and leads to
protonated analytes. Provided that these ions are not
neutralized by absorption of photoelectrons or electrons
from the metallic target [18, 55, 56], they can be detected as
so-called Lucky Survivors.
However, the original Lucky Survivor model [17] had
limitations in the description of analyte anion generation.
Clusters with excess negative charges can quantitatively
neutralize positively precharged analytes, but subsequent
analyte deprotonation by counterion neutralization will be
endergonic because the carboxylic acid groups of
peptides and proteins are less acidic than common
counterions such as trifluoroacetate or chloride. To
account for this a refined Lucky Survivor model was
developed [50], which postulates that matrix excess
charges [ma + H]+ or [ma – H]– included in the clusters
lead to generation of protonated analytes by counterion
neutralization or to analyte deprotonation, respectively.
Matrix anions generally consist of carboxylates or
delocalized phenolates whose neutralized counterparts
exhibit about the same acidity as the analyte carboxylic
acid groups. Consequently, analyte deprotonation should
not be very efficient, which is in agreement with the
typically low intense anionic analyte signals achievable
using acidic matrices [26, 49].
Both models, the refined Lucky Survivor model as well
as the gas phase protonation model, comprise the necessity
of protonated matrix ions [ma + H]+ for analyte protonation.
This is in agreement with the results of Land and Kinsel
[14], who proved a correlation between the matrix radical
cation intensity as assumed precursor species for [ma + H]+
according to Equation 1, [5] and the intensity of protonated
analytes.
maꢀþ þ ma ! ½ma ꢁ Hꢂꢀ þ ½ma þ Hꢂþ
ð1Þ
In addition, quenching of matrix ions at the expense of
analyte protonation (also called the “matrix suppression
effect” [15, 57]) could be detected by means of angular
and time resolved intensity distributions of matrix and
analyte ions [6, 7]. However, despite numerous inves-
tigations and indications regarding the analyte protona-
tion, the final elucidation of the actual mechanism is still
missing.
Experimental
Materials and Reagents
α-Cyano-4-hydroxycinnamic acid (CHCA [58]) was pur-
chased from Bruker Daltonics (Bremen, Germany), acetoni-
trile (ACN, Rotisolv HPLC gradient grade) was from Roth
(Karlsruhe, Germany), and p-anisaldehyde (99+%) was
obtained from Acros (Nidderau, Germany). MilliQ water
was prepared by a Millipore (Schwalbach, Germany) water
purification system. The peptides ITITPNK and ITITPNV
were obtained from the Central Peptide Synthesis Unit of the
German Cancer Research Center (DKFZ). All other chem-
icals were purchased from Sigma-Aldrich (Taufkirchen,
Germany) and were of the highest grade available.
Synthesis of Undeuterated and Deuterated Matrix
Esters
In a first step, cyanoacetic acid was esterified to cyanoacetic
acid-tert-butylester-d9 according to the synthesis route given
by Neises and Steglich [59]. Briefly, 1 eq cyanoacetic acid
was dissolved in sufficient amounts of dry CH2Cl2/ACN
(1:3, vol/vol). Upon addition of 0.07 eq 4-dimethylamino-
pyridine and 1.25 eq tert-butanol-d10, the reaction mixture
was cooled to 0 °C and 1.1 eq dicyclohexylcarbodiimid were
added at small portions. After stirring for 5 min, the ice was
removed and the reaction was stirred further 20 min at room
temperature. After removing solid byproducts by filtration,
the solvent was evaporated and the crude tert-butylcyanoa-
cetate-d9 was purified by short-path distillation at 12 mbar
and 84 °C. Yield: 68% of the theoretical value as clear oil.
For subsequent syntheses of the α-cyanocinnamic acid ester
derivatives, Knoevenagel condensation reactions using the
The Gas Phase Protonation Model
This model predicts neutral analytes in the gas phase
originating either from incorporation in the matrix crystals
as uncharged species or from quantitative charge recombi-
nation with the respective counterions in the case of
precharged analytes. Gas phase collisions of neutral analytes
with protonated [ma + H]+ or deprotonated [ma – H]– matrix

978
T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
deuterated or undeuterated cyanoacetic acid esters and the
respective benzaldehyde (4-hydroxybenzaldehyde or 4-
chlorobenzaldehyde) were performed according to the
instructions given in the literature [60]. Yields for the
cyanocinnamic acid ester syntheses were between 82%–
95% as white (4-chloro-α-cyanocinnamic acid (ClCCA
[28])-ester) to yellow (CHCA-esters) crystals.
parameter settings for standard MS measurements were
chosen and were polarity, positive; operation mode, reflec-
tor; accelerating voltage, 20 kV; grid voltage, 70%; low
mass gate, off; delay time, 150 ns; shots per spectrum, 500;
time resolution, 0.5 ns. For the peptides ITITPNK and
ITITPNV, the grid voltage was set to 68.5% and the low
mass gate to 550 Da. Data Explorer 4.5 (Applied Bio-
systems, Darmstadt, Germany) was used for analysis of the
spectra. Peak detection was performed by using a S/N
threshold of 10 after external calibration and advanced
baseline correction with preset parameters. The chosen
parameters for the MS/MS fragmentation of the CHCA-
tert-butylester were as follows: laser fluence, 1.2 μJ/pulse;
polarity, positive; operation mode, reflector; accelerating
voltage, 20 kV; grid voltage, 68.5%; delay time, 150 ns; low
mass gate, off; bin size, 0.5 ns; shots per segment, 500;
precursor mass, 246 Da; post-source decay (PSD) mirror
ratios, 1, 0.8, 0.75, and 0.5; guide wire for all segments,
0.05%.
The CHCA-tert-butylester-d9 degree of deuteration was
determined using a modified ESI orthogonal time-of-flight
mass analyzer (Mariner; Applied Biosystems, Framingham,
MA, USA) equipped with a nanoelectrospray source and a
heated transfer capillary. The mass spectra of the deuterated
ester (2 mM in 80% ACN/0.1% TFA) were acquired in
positive ion polarity for one minute at a spray potential of
800 V and a nozzle potential of 50 V. The temperature of the
transfer capillary was set to 180 °C.
Sample Preparation and Measurement
Polished stainless steel substrates and standard dried droplet
preparations were used for all experiments. For the MS and
MS/MS matrix spectra, 0.5 μL of matrix solution (20 mM,
70% ACN) were spotted onto a target and air dried. For
experiments applying analytes, mixtures of 0.5 μL of the
matrix solution (20 mM, 70% ACN) and 1 μL of the analyte
solution (10 μM, 30% ACN/0.1% TFA in the cases of the
peptides ITITPNK and ITITPNV, 100 μM in all other cases)
were spotted without premixing onto a target and air dried.
Possible additions of acid or base are indicated at the
appropriate places in the text. The reason for the comparably
high analyte amounts required for the neutral matrix esters is
assumed to be the lacking ion-pair formation possibility
between deprotonated matrix and protonated analyte ions,
which is hypothesized to be crucial for effective analyte
incorporation [28].
Mass Spectrometry
All MALDI MS measurements were carried out using a
standard Voyager DE-STR TOF mass spectrometer (Applied
Biosystems, Darmstadt, Germany) with a VSL-33737ND
337 nm nitrogen laser (LSI Laser Science, Newton, MA,
USA) producing 3 ns laser pulses with a pulse repetition rate
of 20 Hz and an approximately circular spot size of 50–
100 μm in diameter. For pure CHCA as well as undeuterated
and deuterated CHCA- and ClCCA-ester spectra, 10 single
mass spectra with 500 laser shots each were accumulated.
Also, for the experiments using the glycine monomer, dimer,
and hexamer as well as the peptides ITITPNK and ITITPNV
as analytes, all measurements were repeated 10-fold with
500 laser shots each. For all other mass spectra, four mass
spectra with 500 laser shots each were accumulated. Laser
fluences optimized for highest analyte S/N ratios were used
for all approaches comprising analytes in which the absolute
energy is not indicated. The optimum pulse energies were
determined to about 1.5 μJ/pulse for CHCA, undeuterated
and deuterated CHCA-tert-butylester and to about 1.9 μJ/
pulse for ClCCA-tert-butylester-d9 using 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. For
analyzing pure matrices, the laser fluences were lowered by
about 0.4 μJ for all used compounds, which were still about
10% above the respective threshold. Typical MALDI-TOF
Results and Discussion
Discrimination Between Lucky Survivor and Gas
Phase Protonation
Discrimination between the Lucky Survivor and gas phase
protonation theory should be possible provided the matrix
only delivers deuteronated matrix ions [ma + D]+ for gas
phase ionization processes. According to the gas phase
protonation model, this will lead to deuteronation instead of
protonation of the uncharged analyte A (see Scheme 1, first
row). In case of the validity of the Lucky Survivor model
n+
and positively precharged analytes AHn with the corre-
sponding counterions nX–, protonated analytes can be
expected also in presence of deuteronated matrix ions either
by incomplete charge neutralization during cluster gen-
eration and/or cluster decomposition or as a result of
counterion neutralization by [ma + D]+ leading to the
generation of neutral DX (see Scheme 1, second and
third row). Therefore, validation of the proposed models
would be directly possible by screening the mass spectra
for the presence of either [A + H]+ or [A + D]+.
For this purpose, a deuterated matrix compound, which
enables exclusive generation of [ma + D]+ is necessary.
According to Equation 1, generation of [ma + D]+ requires
transfer of a D+ from a deuterated matrix radical cation ma•+
to a neutral matrix molecule. Polarized bonds as present in,

T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
979
+ [ma+D]+
gas phase protonation model
AD+
analyte deuteration
A
(uncharged analyte)
- ma
counterion separation
(original model)
X-
+ n-1HX
n+
n+
X-
X-
AH+ +
AHn
+
+
n
Lucky Survivor model
(precharged analyte)
+ [ma+D]+
- ma
counterion neutralization
(refined model)
AH+ + DX + n-1HX
AHn
n
Scheme 1.
e.g., deuterated hydroxyl- (–OD) or carboxylic acid
(–COOD) groups will strongly alleviate bond breaking with
transfer of D+ compared with nonpolar C–D bonds. There-
fore, pronounced [ma + D]+ generation necessitates matrix
compounds with, e.g., deuterated hydroxyl- and/or carbox-
ylic acid groups. However, such deuterations are instable
during conventional dried droplet (DD) sample preparation
due to D/H-exchange reactions in protic solvents, with
analyte functionalities or also the humidity of the air. The
use of deuterated solvents for avoiding loss of matrix
deuteration will cause at least partial H/D exchanges of the
analyte which has to be prevented since in this case the
origin of analyte deuteration cannot be unequivocally
determined. Consequently, the strategy was to synthesize
deuterated matrix compounds without solution-phase D/H-
exchange by using stable C–D bonds. Upon excitation by
laser irradiation, the matrix to synthesize has to undergo a
rearrangement leading to the generation of polarized –OD or
–COOD groups for subsequent generation of [ma + D]+.
signals can also be detected for all other investigated esters.
This suggests that the ester fragmentation is not finished
until acceleration voltage is applied but already begins in the
very early phase of the expanding plume. For more detailed
discussion concerning the matrix ester fragmentation see SI
Data 1.
For generation of the deuterated acid CHCA-COOD
and its deuteronated counterpart [CHCA-COOD + D]+
the CHCA-tert-butylester-d9 (MWneutral,
=
monoisotopic
254.16 g/mol) was synthesized and its mass spectrum was
compared with that of the undeuterated CHCA-tert-butylester
(MWneutral, monoisotopic=245.10 g/mol), see Figure 1. In
Figure 1a, the protonated CHCA-tert-butylester and the
corresponding fragment ion, the protonated CHCA acid at
m/z=190.05, can be detected. Figure 1b refers to the deuterated
ester and exhibits a signal at m/z=256.18, which correlates to
the deuteronated ester [CHCA-tert-butylester-d9 + D]+ and its
fragment ion at m/z=192.06 representing the desired deuter-
ated and deuteronated free acid [CHCA-COOD + D]+. A small
amount of the d8-compound is indicated by the low-intensity
signals at m/z=255.17 and m/z=191.06 in Figure 1b and is
caused by incomplete deuteration of the tert-butyl group as
verified by ESI-MS (SI Figure 3). The deuteronated ester
[CHCA-tert-butylester-d9 + D]+ should also be able to
deuteronate analytes although its reactivity is somewhat
lowered compared with the deuteronated acid. Regarding
the discussed need for exclusive generation of deutero-
nated matrix ions to verify gas phase analyte deuterona-
tion, also the [CHCA-tert-butylester-d9 + D]+ fulfills this
necessity.
The only source of deuterons for generation of the
deuteronated tert-butylester-d9 and subsequently for the
deuteronated acid [CHCA-COOD + D]+ is the deuterated
ester group. For energetic reasons, it could be expected that
the CHCA-tert-butylester-d9 radical cation transfers a proton
from the 4-hydroxyl group instead of a deuteron from a
more stable C–D bond of the ester group. Nevertheless,
deuteron abstraction could cause a subsequent loss of
isobutene-d8 and generation of [CHCA – H]• according to
Equation 1. Although the exact reasons are not known in
detail, it can be assumed that abstraction of a deuteron is
energetically favored due to entropy and/or that the larger
surface of the tert-butyl group compared with the hydroxyl
group increases the probability for deuteron abstraction
instead of that of a proton. Consequently, the missing
A Matrix for Exclusive Generation
of the Deuteronated Matrix Species [ma + D]+
From experiments carried out earlier in the systematic
investigation of the role of functional groups on matrix
properties of α-cyanocinnamic acid derivatives [28], we
recollected that matrix esters typically yield two prominent
ion signals, which are the protonated intact molecule and the
protonated free acid as a fragment ion. Fragmentation of the
ester to the acid can be proven by PSD analysis of the
protonated ester, which yields nearly exclusive generation of
the protonated free CHCA acid (compare Supplemental
Information (SI) Figure 1 for the PSD spectrum of
protonated CHCA-tert-butylester). It was tested whether a
matrix ester, which is per-deuterated at the ester group,
behaves in the expected way, thus forming the discriminat-
ing test reagent matrix needed. A possible contamination by
the free matrix acid as a synthesis byproduct can be easily
detected or excluded by the inspection of the ester mass
spectra since present free acids will produce radical
molecular ions, see SI Figure 2. The low-resolved signal
shifted to a somewhat higher m/z-range in SI Figure 2b
cannot be detected in the upper spectrum of the free acid and
is most probably due to PSD generated matrix acid from the
matrix ester after ion acceleration. Similar low-resolved

980
T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
190.05
100
80
60
40
20
0
10,000
OH
O
CH3
O
C+
[ma+H]+
CH3
CH3
OH
246.11
CN
HO
CN
HO
CHCA-tert-butylester
MWmono = 245.10 g/mol
247.12
249
191.05
185
201
217
233
265
mass (m/z)
(a)
192.06
100
80
60
40
20
0
10,000
OD
C+
O
CD3
O
CD3
CD3
OD
[ma+D]+
CN
256.18
HO
CN
HO
CHCA-tert-butylester-d9
MWmono = 254.16 g/mol
193.07
257.18
191.06
255.17
185
201
217
233
249
265
(b)
mass (m/z)
Figure 1. Positive-ion mode spectrum of 10 nmol (a) CHCA-tert-butylester and (b) CHCA-tert-butylester-d9, respectively.
Identical parameters were used for recording the matrix spectra
detectability of the ester radical cations in the mass spectra
of Figure 1 might be explained by a high efficiency of this
reaction. In order to completely exclude the involvement of
the hydroxyl-hydrogen and to verify the role of matrix
proton affinity [28], ClCCA-tert-butylester-d9 was synthe-
sized as a second deuterated test matrix. It was not
determined whether gas phase protonations and deuterona-
tions exhibit identical reaction rates.
matrix acid [CHCA-COOD + D]+ and the deuteronated analyte
[p-anisaldehyde + D]+ at m/z=138.07 can be detected in
Figure 2b. The given close-ups substantiate either exclusive
analyte protonation or deuteronation according to the postu-
lation of the gas phase protonation model. In agreement with SI
Figure 4 yielding comparable results with 4′-hydroxyaceto-
phenone as neutral analyte, this confirms the correctness of the
gas phase protonation model.
Neutral Analytes A
Medium Basic Analytes A/[A + H]+
In a first case, neutral analytes A were used for investigating
the MALDI analyte protonation mechanism by means of
p-anisaldehyde (p-methoxybenzaldehyde) and 4′-hydroxyace-
tophenone. Matrix effects such as photoionization of the used
analytes can be excluded due to their very low extinction
coefficients at 337 nm and much lower concentrations
compared with the used matrices CHCA-tert-butylester-d9
and its undeuterated counterpart. Since neutral analytes are
incorporated without charges, only the gas phase protonation
model is applicable to explain protonation of neutral analytes.
Provided the correctness of this model, it can be expected that
application of the undeuterated and deuterated CHCA-tert-
butylester matrix cause protonation or deuteronation of the
neutral analytes, respectively. In agreement with this, Figure 2a
shows the protonated acid [CHCA + H]+ generated upon
fragmentation of the undeuterated CHCA-tert-butylester
together with the protonated analyte [p-anisaldehyde + H]+ at
m/z=137.06. Contrary to this, the deuterated and deuteronated
Medium basic analytes can exist as neutral as well as
protonated species depending on the pH of the sample
preparation solution. According to experiments regarding the
incorporation of pH-dependent dyes [52] or incorporation of
stable adducts between protonated analytes and anions of
extremely strong acids [53, 54], the incorporated analytes
maintain their respective solution charge states within the
matrix crystal. Therefore, it is reasonable to assume that
medium basic analytes are incorporated both as neutral and
protonated species in the matrix crystal in dependence of the pH
conditions during crystallization. In a first approach, nicotina-
mide (niacinamide, MWneutral, monoisotopic=122.05 g/mol)
with a pKa value of 3.4 [61] was used as medium basic analyte
together with the undeuterated neutral matrix CHCA-tert-
butylester at neutral pH values, see SI Figure 5. Under these
conditions, the vast majority of nicotinamide exists as
uncharged species, however, a small amount is still protonated.
No acid was added to the preparation solution to avoid influence

T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
981
190.05
100
80
60
40
20
0
137.06
[A+H]+
53,000
100
80
60
40
20
0
[CHCA+H]+
2,978
close-up
191.05
OH
C+
138.06
H
133
135
137
139
141
143
mass (m/z)
172.04
MeO
155.04
158
137.06
130
144
172
186
200
(a)
mass (m/z)
192.06
[CHCA+D]+
100
80
60
40
20
0
138.07
100
80
60
40
20
0
32,000
2,323
[A+D]+
close-up
OD
C+
139.07
139
193.07
H
133
135
137
141
143
mass (m/z)
MeO
155.05
191.06
172.04
138.07
130
144
158
172
186
200
mass (m/z)
(b)
Figure 2. Protonation and deuteronation of the neutral analyte p-anisaldehyde (MW=136.15 g/mol) using the matrix (a)
CHCA-tert-butylester or (b) CHCA-tert-butylester-d9, respectively. Please remember that the monoisotopic peaks of [CHCA-
COOH + H]+ and [CHCA-COOD + D]+ are saturated in some of the single spectra accumulated for Fig. 2. The insets show a
close-up of the analyte m/z range. Identical parameters were used for recording the spectra
of the neutral-to-protonated analyte ratio A/[A + H]+. As
can be seen in SI Figure 5a, nicotinamide can be detected
exclusively as protonated species at m/z=123.06 together
with the protonated acid [CHCA-COOH + H]+ generated
upon fragmentation of the undeuterated CHCA-tert-buty-
lester. Subsequently, nicotinamide was used together with
the deuterated matrix ClCCA-tert-butylester-d9 for SI
Figure 5b. In this case, not only the protonated but also the
deuteronated nicotinamide can be detected at m/z=123.06 and
124.06, respectively. Additionally, the deuterated and deutero-
nated acid [ClCCA-COOD + D]+ is detected at m/z=210.03
and 212.03 as the main fragment ion of the deuterated ClCCA-
tert-butylester-d9. Generation of the protonated as well as
deuteronated nicotinamide can be explained by Scheme 2:
Both neutral and protonated nicotinamide is incorporated
in the matrix crystals. In accordance with previously
discussed deuteronation of neutral analytes, the neutral
portion of nicotinamide can undergo gas phase deutero-
nation to [A + D]+ according to the gas phase protonation
model. However, the protonated nicotinamide percentage
originating from the preparation solution gets detectable
as [A + H]+ upon anion neutralization or -separation
corresponding to the Lucky Survivor model. Application of
the undeuterated CHCA-tert-butylester only leads to the
exclusive generation of protonated nicotinamide [A + H]+
although two different ionization pathways are involved.
The reason for using the ClCCA-tert-butylester-d9
instead of the CHCA-analog was to avoid the possibility of
D/H-exchange reactions between deuteronated nicotinamide
[A+D]+
[A+H]+
Lucky
Survivor
gas phase
deuteronation
[ma+D]+
O
O
NH2
NH2
+
N+
H
N
gas phase
protonation
Lucky
Survivor
[ma+H]+
[A+H]+
[A+H]+
Scheme 2.

982
T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
[A + D]+ and the 4-hydroxyl group of the CHCA matrix as
alternative explanation for generation of the protonated
nicotinamide. Although such D/H-exchanges could not be
detected for the previously discussed deuteronated analytes
p-anisaldehyde or 4′-hydroxyacetophenone using CHCA-
tert-butylester-d9, application of ClCCA-tert-butylester-d9
clearly proves the existence of the Lucky Survivor process
since no acidic hydrogens are available in ClCCA-tert-
butylester-d9 or [ClCCA-COOD + D]+ capable of explain-
ing an alternative generation of [A + H]+.
Simultaneous detection of protonated and deuteronated
medium basic analytes by means of parallel Lucky Survivor
and gas phase protonation or deuteronation in the case of
deuterated matrices, respectively, allows for investigation of
the parameters influencing each ionization pathway. Varia-
tion of the pH value during sample preparation leads to
increased or decreased amounts of protonated analytes and
therefore to a promotion or suppression of the Lucky
Survivor process. Figure 3a demonstrates the influence of
pH-variation for the medium basic analyte nicotinamide and
the deuterated matrix ClCCA-tert-butylester-d9 (top: addi-
tion of one μL 0.1 M NaOH and bottom: one μL 1% TFA
solution to the DD preparation, respectively). No radical
cations of the corresponding matrix acids could be detected
in the mass spectra upon base or acid addition thereby
proving that no ester cleavage occurred in solution during
sample preparation. With decreasing pH value, the percent-
age of nicotinamide incorporated in the matrix crystals as
protonated species increases, leading to an activation of the
Lucky Survivor and to an increase of [A + H]+ at m/z=
123.06 compared with [A + D]+ originating from gas phase
deuteronation, see Figure 3a.
Activation of the gas phase protonation process requires
higher amounts of the protonating matrix species [ma + H]+
or [ma + D]+ in the case of analyte deuteronation,
respectively. According to Equation 1, the yield of these
matrix ions depends on the amount of matrix radical cations,
which in turn depends on the applied laser fluence. Increase of
the laser fluence, consequently, activates gas phase deuterona-
tion as is verified by the mass spectra of Figure 3b, which are
124.06
AD+
124.06
AD+
100
80
60
40
20
0
100
1,456
50,000
fluence =
1.7 µJ
basic
80
123.06
AH+
60
40
125.06
AH+
20
0
123.06
110
116
122
128
134
140
1,600
110
116
122
123.06
AH+
128
134
140
9,400
mass (m/z)
mass (m/z)
124.06
100
80
100
AD+
fluence =
1.5 µJ
neutral
124.06
AD+
80
60
40
gas phase
60
Lucky Survivor
deuteronation
40
123.06
AH+
20
0
20
0
125.06
125.06
128
110
116
122
128
134
140
1,304
110
116
122
123.06
AH+
134
140
1,304
mass (m/z)
mass (m/z)
123.06
AH+
100
80
60
40
20
0
100
80
60
40
20
0
fluence =
1.4 µJ
acidic
124.06
AD+
124.06
AD+
116
122
128
134
140
116
122
128
134
140
110
110
mass (m/z)
mass (m/z)
(a)
(b)
Figure 3. Promotion and suppression of Lucky Survivor and gas phase protonation, respectively. (a) Influence of the analyte
protonation state using different solvent pH values at a laser fluence of 1.4 μJ. (b) Influence of the laser fluence at acidic
conditions. ClCCA-tert-butylester-d9 and nicotinamide have been used as matrix and analyte, respectively. One μL 1% (vol/vol)
TFA or 1 μL 0.1 M NaOH solution were added to the DD-preparation for acidic or basic conditions

T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
983
all recorded at acidic conditions. With increased laser fluence
going along with stronger formation of [ma + D]+ ions, an
increase in deuteronated nicotinamide [A + D]+ is observed.
The absolute intensity of [A + H]+ also increases with rising
laser fluence, which can be traced back to a stronger
ablation of matrix material with embedded precharged
analytes [A + H]+ leading to a more intense counterion
separation and/or to a more efficient counterion neutral-
ization of [A + H]+ by [ma + D]+. Thus, also the
contribution of the Lucky Survivor mechanism increases
upon higher laser fluences, although the gain is less
pronounced than in the case of gas phase protonation or
deuteronation.
protonation process is the dominant ionization pathway for
these analyte classes.
For this purpose, the basic peptide ITITPNK (pI=10.1
[62], Figure 4) and the nearly neutral peptide ITITPNV (pI=6
[62], SI Figure 6) were used as analytes and measured with the
undeuterated matrix CHCA-tert-butylester and the deuterated
matrices CHCA-tert-butylester-d9 as well as ClCCA-tert-
butylester-d9. For mimicking typically used acidic preparation
conditions and matrices, 0.1% TFA was added to the
preparation solutions. The detected differences in absolute
ion intensities between CHCA-tert-butylester and its deuter-
ated counterpart are most probably due to different crystal
morphologies as all parameter settings, including laser fluence
and matrix and analyte amounts, were identical. In the case of
the undeuterated CHCA-tert-butylester both analyte ionization
pathways result in generation of [A + H]+ without differ-
entiation possibility, see Figure 4a and SI Figure 6a. The insets
show the calculated isotope ratios of the respective protonated
analytes, which are in good agreement with the measured ones.
At typically used preparation conditions comprising acidic
matrices and addition of, e.g., 0.1% TFA, even acidic peptides
and proteins are nearly quantitatively protonated in solution.
Using the deuterated matrix CHCA-tert-butylester-d9, strong
signals of the protonated analytes [A + H]+ can still be detected
which are in agreement with the Lucky Survivor process, see
We therefore conclude that the nature of the analyte
(basicity) as well as the preparation and measurement
conditions (pH and laser fluence) determines the dominant
analyte protonation pathway which, however, is not dis-
cernible in normal MALDI mass spectrometric experiments.
Basic (Positively Precharged) Analytes [A + H]+
The overwhelming majority of analytes investigated with
MALDI MS are peptides and proteins. It was therefore of
interest whether the Lucky Survivor or the gas phase
AH+
786.472
786.47
100
80
60
100
O
1,513
CH3
calculated isotope
CH3
CH3
80
ratios of AH+
O
AH+, first isotope
60
CN
CHCA-tert-butylester
783.4
787.475
787.48
HO
40
40
20
0
20
788.48
789.49
790.2
0
786
788
790
780.0
786.8
797.0
mass (m/z)
mass (m/z)
(a)
AH+
786.47
100
AD+
O
8,756
CD3
CD3
CD3
80
787.48
O
AH+, first isotope
60
CN
HO
40
788.48
CHCA-tert-butylester-d9
20
0
789.49
790.49
783.4
786.8
790.2
793.6
797.0
(b) 780.0
mass (m/z)
AD+
787.48
100
80
60
40
20
0
O
3,011
CD3
AH+
CD3
AH+, first isotope
786.47
O
CD3
788.48
CN
Cl
789.49
790.49
ClCCA-tert-butylester-d9
791.50
780.0
783.4
786.8
790.2
793.6
797.0
(c)
mass (m/z)
Figure 4. Intensities of the protonated and deuteronated signals of the peptide ITITPNK abbreviated as “A” by using the (a)
undeuterated and (b) deuterated CHCA-tert-butylester as well as (c) the more reactive deuterated ClCCA-derivative. Inset:
calculated isotope ratios of protonated ITITPNK. The horizontal lines represent the isotope intensities of AH+. Identical
parameters were used for recording the analyte spectra

984
T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
Figure 4b and SI Figure 6b. However, the isotopic patterns
reveal an increase in intensity of the isotope peaks marked by
horizontal lines due to overlapping deuteronated analytes [A +
D]+. Therefore, both analyte ionization processes, Lucky
Survivor and gas phase protonation (noticeable as deuterona-
tion), occur. In the case of the CHCA matrix, the vast
majority of the ionized analytes are generated according to
the Lucky Survivor model, which is responsible for about
90% of the overall analyte ions calculated by means of the
respective peak areas. Using the ClCCA matrix with a
lower proton affinity [28, 63–65] results in a more efficient
proton transfer reaction of [ma + H]+ or deuteron transfer
reaction in the case of [ma + D]+ to the neutral analyte, see
Figure 4c and SI Figure 6c. The observation also proves
that the ratio between Lucky Survivor and gas phase
protonation is strongly affected by the matrix proton
affinity. The lower the proton affinity of the matrix, the
higher is the percentage of the gas phase protonation of the
overall analyte ionization.
One can argue that according to line 3 of Scheme 1, a
more reactive matrix with a higher proton and deuteron
transfer efficiency should also favor counterion neutraliza-
tion of the precharged analyte [A + H]+ leading not only to
an increase in gas phase protonation but also of the Lucky
Survivor process. Although this might be true for anions of
very strong acids, neutralization of normally involved anions
such as, e.g., chloride, trifluoroacetate, or matrix anions is
strongly exoergonic, also taking into account an energy barrier
to overcome due to coulomb forces between the charges [54].
Consequently, more reactive matrices should not distinctively
enhance such processes. The detectability of deuteronated
analytes [A + D]+ raises the question of the origin of the neutral
analytes required for this purpose. At acidic pH values, the
basic peptide ITITPNK with a pI of 10.1 exists nearly
quantitatively as protonated species in solution. Therefore,
the detected [A + D]+ intensity cannot be due to deuteronation
of the remaining neutral analytes but can only be explained by
deuteronation of the pool of neutral analytes generated upon
neutralization of precharged [A + H]+. This possibility will be
elucidated in the following.
spectra in Figure 5a, the three analytes were investigated
using the undeuterated matrix CHCA-tert-butylester. At
these conditions, only the protonated analytes [A + H]+ are
generated with their respective isotope patterns. In the case
the deuterated matrix counterpart CHCA-tert-butylester-d9
is used, the expected protonated and deuteronated analytes
can be detected, as shown in Figure 5b. This was already
demonstrated for the peptides ITITPNK and ITITPNV and
can be explained by parallel Lucky Survivor and gas phase
deuteronation processes. A comparison of the deuteronation
efficiencies of the three analytes by means of the respective
[A + D]+/[A + H]+ ratios reveals that with increasing peptide
length the intensity of the deuteronated analytes drops. In the
case of the glycine hexamer, the first isotope peak at
m/z=362.15 predominantly is composed of the 13C-isotope
peak of [A + H]+ and only to a small amount of [A + D]+. At
a first glance, this might be surprising since the analyte
collision cross sections rise with increasing length. Together
with an also increasing number of protonable amide backbone
functionalities one could expect more efficient [A + D]+
generation. The proton affinities of both glycine amino
terminus and amide backbone are very close to each other
(886.2 kJ/mol and 889.9 kJ/mol, respectively [39, 43]) and
are much higher than that of CHCA with 841 kJ/mol [35].
Consequently, the basicity of the respective proton acceptor
groups cannot be responsible for the detected differences in
[A + D]+ generation. It is known that the proton affinity rises
with increasing peptide length due to intramolecular stabiliza-
tion by charge self-solvation [66, 67], which leads to clearly
higher proton affinities for the larger glycine analytes
compared with the glycine monomer [39, 40]. Therefore,
intramolecular stabilizations would cause more intense
[A + D]+ generation for the larger glycine analytes, which
was not observed. This can be explained by the fact that
rearrangement timescales are in the range of ≈100 ns
(α-helices) to ≈6 μs (β-turns) for secondary structure
formation [68–71] and about 50–100 μs for tertiary contacts
[69, 72]. Thus, they are much too slow for influencing the gas
phase protonation/deuteronation process, which happens
within the first few tens of nanoseconds after laser irradiation,
while most of the collisions between matrix and analyte
neutrals and ions occur as is obvious by analyte ion
generation in the linear ion mode without delayed extraction.
Therefore, the only reasonable explanation for the
decreasing [A + D]+ amounts in the cases of the glycine
dimer and hexamer are likewise decreasing amounts of the
corresponding deuteronable neutral analytes. As was dis-
cussed for the gas phase, intramolecular peptide and protein
folding upon analyte protonation during incorporation in the
matrix crystal or already in solution can stabilize the
protonated ions. Consequently, more stable [A + H]+ ions
such as the protonated glycine hexamer will have a higher
probability to survive, which means they should undergo a
more efficient Lucky Survivor process compared with less
stable [glycine + H]+. Therefore, the neutralized analyte
amounts of the glycine dimer and especially hexamer will be
Neutralization of Precharged Analytes
According to the postulation of the Lucky Survivor model,
the predominant process of analytes incorporated in the
matrix crystals as precharged ions should be their neutral-
ization for energetic reasons. Only the fractions that
experience incomplete neutralization can remain as detect-
able ions. To verify this neutralization process as well as to
investigate the possible influence of the analyte structure, the
glycine monomer (MWneutral, monoisotopic=75.03 g/mol),
dimer (MWneutral, monoisotopic=132.05 g/mol), and hexamer
(MWneutral, monoisotopic=360.14 g/mol) were used as analytes.
All three analytes exhibit an isoelectric point of pI=6 [62]
and were, thus, nearly quantitatively protonated upon
addition of 0.1% TFA to the sample solutions. For the

T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
985
CHCA-tert-butylester-d9
CHCA-tert-butylester
77.05
76.04
Gly+H+
100
80
60
40
20
0
100
80
60
40
20
0
Gly+D+
glycine1
1,423
969
76.04
78.05
72.0
74.2
76.4
mass (m/z)
78.6
80.8
83.0
72.0
74.2
76.4
mass (m/z)
78.6
80.8
83.0
133.06
Gly2+H+
133.06
100
80
60
40
20
0
100
80
60
40
20
0
1,902
2,278
glycine2
134.07
Gly2+D+
135.07
134.12
136.08
137
135.08
129
131
133
135
mass (m/z)
137
139
129
131
133
135
mass (m/z)
139
361.15
Gly6+H+
361.15
100
80
60
40
20
0
100
80
60
40
20
0
872
1,389
glycine6
Gly6+H+, first isotope
(Gly6+D+)
362.15
362.15
363.15
363.4
363.15
364.16
358.0
359.8
361.6
mass (m/z)
365.2
367.0
358.0
359.8
361.6
363.4
365.2
367.0
mass (m/z)
(a)
(b)
Figure 5. Isotopic patterns of the protonated glycine monomer, dimer, and hexamer using (a) the undeuterated matrix CHCA-
tert-butylester and intensities of the protonated and deuteronated signals of the glycine monomer, dimer, and hexamer using
(b) the deuterated matrix CHCA-tert-butylester-d9. The low-resolved signal in the deuterated glycine dimer spectrum refers to a
CHCA-tert-butylester-d9 fragment generated by PSD fragmentation
lower, which results in only low gas phase deuteronation
intensities. Vice versa, less stable protonated glycine under-
goes a more efficient neutralization leading to a higher
probability for subsequent gas phase deuteronation.
In summary and in addition to the effects discussed in the
chapter “Medium Basic Analytes A / [A + H]+”, the
efficiency of the Lucky Survivor further depends on the
ability of the precharged analytes to stabilize their charge
and therefore on their respective size and composition. This
reflects the common experience that larger [73] and more
basic analytes [74–76] exhibit higher ion yields.
acids generated upon ester cleavage seem to be able to
generate [A + D]+. Gas phase backbone H/D exchanges can
lead to multiply deuterated analytes, causing broadening or
complete shifting of the isotope pattern [77–79]. Never-
theless, the vast majority of the deuteronated analytes exhibit
attachment of only one deuteron, which does not reflect the
possibility of multiple H/D exchanges but is in accordance
with the result of gas phase deuteronations (compare
Figures 3, 4 and 5). Additionally, no D/H exchanges have
been observed in the case of the deuteronated analytes
p-anisaldehyde and 4′-hydroxyacetophenone with the hydro-
gen of the CHCA-tert-butylester-d9 hydroxyl group which
contradicts efficient H/D-plume exchanges, see Figure 2 and
SI Figure 4. Application of ClCCA-tert-butylester-d9 and its
deuteronated fragmentation product [ClCCA-COOD + D]+
increase the analyte deuteronation efficiency compared with
the CHCA-counterpart, as shown in Figure 4 and SI Figure 6.
This cannot be explained by more efficient H/D-exchanges
of protonated analytes since ClCCA is more acidic than
CHCA [28] and the exchange rates decrease with reagent
acidity [77, 80]. It is, however, in accordance with a higher
deuteron transfer capability due to the lower proton affinity
of ClCCA [28, 60, 63]. Further, taking into account that
Alternative Explanation Possibilities
for Generation of Deuteronated Analytes [A + D]+
Deuteronation of the neutral analytes p-anisaldehyde and 4′-
hydroxyacetophenone clearly proves the existence of gas
phase deuteronation. However, H/D-exchange reactions of
[A + H]+ with deuterated matrices might also account for
generation of [A + D]+. Conversion of [A + H]+ to [A + D]+
upon collision with neutral deuterated matrix esters seems to
be improbable because of their stable C–D bonds. Therefore,
only collisions of [A + H]+ with neutral deuterated matrix

986
T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
glycine hexamers produce less [A + D]+ than the shorter
glycine analytes, even though they exhibit a larger
collision cross-section and, therefore, should undergo
more efficient H/D-exchanges, it can be concluded that
the amount of [A + D]+ generated upon H/D exchanges
of protonated analytes is low.
As emphasized in the chapter “Neutralization of Pre-
charged Analytes,” the proton affinities of the amino-
terminus and of the amide backbone as least basic proton
acceptors are still much higher than that of common
matrices. Therefore, subsequent neutralizations of proto-
nated analytes are possible only with deprotonated ions such
as, e.g., [ma – H]–. Consequently, it appears unlikely that the
generation of deprotonated analyte ions [A – H]– occurs
according to the Lucky Survivor process of positively
precharged analytes, which necessitates 2-fold analyte
deprotonation with participation of [ma – H]– but rather to
deprotonation of neutral analytes resulting from charge
neutralization upon cluster decay, see Figure 6. The gas
phase basicities of deprotonated α-amino acids [38] are
clearly higher than that of the CHCA matrix anion [35],
which leads to endergonic analyte deprotonation accord-
ing to the equilibrium model for charge transfer between
matrix and analyte ions [5, 44]. In addition to this, the
assumed exclusion of the important Lucky Survivor
pathway for [A – H]– generation further substantiates
the positive/negative analyte ion yield ratio 91 typically
achieved with acidic matrices [26, 49].
The Unified MALDI Analyte Protonation
Mechanism
Based on previously discussed results, which proved the
correctness of the Lucky Survivor as well as the gas phase
protonation process, a unified MALDI analyte protonation
mechanism was created. A simplified version with restric-
tion to positively precharged analyte ions such as peptides
and proteins is given in Figure 6. According to the Lucky
Survivor model, positively charged clusters release proto-
nated analyte ions [A + H]+ upon dissociation either by
lacking anions of preformed analyte ions or more likely by
anion neutralization with participation of additionally incor-
porated [ma + H]+. The surviving analyte charge originates
from solution and is, therefore, initially localized on a basic
analyte functional group, which was protonated during
sample preparation. Subsequent liberation to the amide
backbone according to the mobile proton model becomes
possible after ion activation [81–83]. In the case of net
neutral clusters, complete charge neutralization occurs upon
dissociation releasing neutral A. Proton transfer reactions
from [ma + H]+ can protonate the analytes according to the
gas phase protonation model. In dependence on the analyte
composition and conformation, amide backbones exhibit the
highest probability for direct proton attachment. Which of
the two protonation pathways dominates depends on
parameters such as, e.g., laser fluence, matrix (and analyte)
proton affinity, analyte composition and size, and the
protonated-to-neutral analyte ratio [A + H]+/A embedded
in the matrix crystal.
Conclusions
The two long since postulated MALDI analyte protonation
pathways known as the Lucky Survivor and the gas phase
protonation model could both finally be experimentally
proven. Several parameters favoring either the Lucky
Survivor or the gas phase protonation were detected and
investigated in detail. In addition to laser fluence, matrix and
analyte proton affinity and the protonated-to-neutral analyte
ratio embedded in the matrix crystal also the analyte
composition and size affects the protonation pathway. With
increasing size, the investigated peptides were preferably
protonated according to the Lucky Survivor mechanism. We
ascribe this to more pronounced intramolecular foldings of
larger analytes during incorporation in the matrix crystal or
already in solution going along with higher survival
probabilities of the intramolecular solvated precharged
species. It can be expected that for the same reason the
survival probabilities of larger precharged peptides and
proteins are even higher. In the case of the commonly used
matrix CHCA or its deuterated counterpart, respectively, the
investigated peptide analytes were ionized to about 90%
according to the Lucky Survivor process. However, the
efficiency of the gas phase protonation process strongly
increases by using more reactive matrices with lower proton
affinities such as ClCCA. Under these conditions, both
protonation pathways exhibited nearly same probabilities.
The detection of protonated analyte ions in cases of
deuterated matrices without any exchangeable hydrogens
confirmed the previously claimed incorporation of analytes
in their respective solution charge states [52–54].
Lucky Survivor
+ maH+
AH+
basic site
- ma - nHX
charge mobilization
cluster
decay
AHnn+ nX-
AH+
amide backbone
+ maH+
- ma
+ [ma-H]-
- ma
gas phase
protonation
matrix cluster with
preformed analyte ion
A
- nHX
+ maH+
- ma
+ [ma-H]-
- ma
[A-H]-
Figure 6. The Unified MALDI Analyte Protonation Mecha-
nism simplified to positively precharged analyte ions. The
mechanism combines and connects the Lucky Survivor and
the gas phase protonation model
In a way, it could be expected that both postulated
ionization pathways are true, taking into account the multi-

T. W. Jaskolla and M. Karas: The Unified MALDI Analyte Protonation Mechanism
987
16. Zenobi, R., Knochenmuss, R.: Ion formation in MALDI mass
spectrometry. Mass Spectrom. Rev. 17, 337–366 (1998)
17. Karas, M., Glückmann, M., Schäfer, J.: Ionization in matrix-assisted
laser desorption/ionization: singly charged molecular ions are the lucky
survivors. J. Mass Spectrom. 35, 1–12 (2000)
18. Frankevich, V., Knochenmuss, R., Zenobi, R.: The origins of electrons
in MALDI and their use for sympathetic cooling of negative ions in
FTICR. Int. J. Mass Spectrom. 220, 11–19 (2002)
19. Breuker, K., Knochenmuss, R., Zhang, J., Stortelder, A., Zenobi, R.:
Thermodynamic control of final ion distributions in MALDI: in-plume
proton transfer reactions. Int. J. Mass Spectrom. 226, 211–222 (2003)
20. Karas, M., Krüger, R.: Ion formation in MALDI: the cluster ionization
mechanism. Chem. Rev. 103, 427–439 (2003)
tude of investigations regarding these models. Consequently,
it stands to reason that both Lucky Survivor and gas phase
protonation are parts of one greater overall mechanism,
which we tried to account for with the presented Unified
MALDI Analyte Protonation Mechanism. Therefore, this
work finally ends a long-lasting discussion about how at
least a partial aspect of one of the most important techniques
in current molecular analysis works.
21. Dreisewerd, K.: The desorption process in MALDI. Chem. Rev. 103,
395–425 (2003)
Acknowledgments
22. Fournier, I., Marinach, C., Tabet, J.C., Bolbach, G.: Irradiation effects
in MALDI, ablation, ion production, and surface modifications. part III.
2, 5-Dihydroxybenzoic acid Monocrystals. J. Am. Soc. Mass Spectrom.
14, 893–899 (2003)
The use of halogenated MALDI matrices is subject of a
German patent and international patent application.
The authors acknowledge financial support from the WI
Bank for economic and public infrastructure development.
23. Syage, J.A.: Mechanism of [M + H]+ formation in photoionization mass
spectrometry. J. Am. Soc. Mass Spectrom. 15, 1521–1533 (2004)
24. Knochenmuss, R.: Ion formation mechanisms in UV-MALDI. Analyst
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25. Meyer, M.A.R., Adams, N., Schubert, U.S.: Statistical approach to
understand MALDI-TOF MS matrices: discovery and evaluation of new
MALDI matrices. Anal. Chem. 79, 863–869 (2007)
26. Dashtiev, M., Wäfler, E., Röhling, U., Gorshkov, M., Hillenkamp,
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