RAPID COMMUNICATIONS IN MASS SPECTROMETRY,VOL.

10,1873-1880(1996)

Matrix-assisted Laser Desorption/Ionization in

Transmission Geometry: Instrumental
Implementation and Mechanistic Implications
Martin Schiirenberg,* Torsten Schulz, Klaus Dreisewerd, Franz Hillenkamp
Institute of Medical Physics and Biophysics, University of MUnster, Robert-Koch-Str.31.48149Miinster. Germanv

Matrix-assisted laser desorption/ionization (MALDb spectra of peptides and proteins in the mass range
1-150 kDa, obtained with the same instrument using both the usual top illumination and also using a novel
transmission illumination geometry are reported. 2,5-Dihydroxybenzoic acid @HB) and a-cyano-rl-hydroxycinnamic acid were used as matrices. In transmission geometry, ions are generated by MALDI rather than
laser-induced pressure pulses, they originate from the irradiated sample face. Spectra obtained with the two
geometries are virtually indistinguishable with only minor differences in threshold fluence, signal-to-noise ratio,
mass resolution and number of successful exposures from a given sample. Spectra were also obtained from
specially mounted cytochrome c-doped single crystals of DHB. Using delayed ion extraction, the initial velocity
of ions upon extraction was measured. In the transmission geometry, the mean initial velocities were zero for
delay times of >0.5 p,in contrast to a mean axial velocity of c a 650 m/s under prompt extraction conditions.
This result is interpreted as a randomization of the directions of the initial velocity distribution, resulting in a
partial or full thermalization of the demrption plume in the conlined space between the crystal bottom face and
the glass substrate. A mathematical analysis of the time-of-fight equation for ions from such thermalid
MALDI-plumes is presented.

Since its introduction in 19Mi matrix-assisted laser desorptionhonization mass spectrometry (MALDI-MS) has
become a standard method for mass spectrometry of
biopolymers (e.g. Ref. 2). In standard configurations of the
ion source, the sample is placed on a metal substrate and
irradiated from the side facing the analyzer (top illumination). In contrast to this, in transmission geometry, the
rear side of the sample is irradiated through a transparent
substrate, either by a focused laser beam or, as described
here, with an optical fiber.
In the transmission mode, geometrical restrictions
imposed by the necessity to guide the laser beam through
the ion optics can be overcome. Transmission geometries
could also become important in the future in combination
with laser diodes. Due to their small size and expected low
cost, laser diodes are, in principle, interesting radiation
sources for MALDI, once powerful diodes with shorter
emission wavelengths or matrices suitable for the longer
wavelength range become available. Diode arrays will,
however have to be used to obtain the necessary energy per
pulse for nanosecond pulses. They have a notoriously low
hrightness, even if pigtail fiber-optical couplers are used,
mile guiding the beam over longer distances with conventional optics and a limited aperture is impossible. Using
transmission geometry, the emitting area of a single diode or
a pigtail diode array could be placed directly behind the rear
surface of a thin transparent target, thereby achieving a
sufficiently small desorption area, despite the high beam
divergence.
To date, very few studies of transmission geometries in
combination with MALDI-MS have been carried out. Vertes
et a1.3 were the first to demonstrate matrix-assisted laser
desorption of small peptides using a LAMMA 500 (Leybold-Heraus) instrument at 266 nm wavelength with
nicotinic acid as a matrix. Lennon and Glish4 likewise
obtained transmission mass spectra of peptides using
Author for correspondence.
CCC 0951-4198/96/15
187348
0 1996 by John Wiley & Sons,Ltd.

a-cyano4hydroxycinnamic acid (aHCCA) as matrix. In

their study, the sample was placed on the front face of a
3 mm long, 2.5 mm diameter quartz cylinder, while the laser
light (A= 355 nm) was coupled into its rear face by means of
an optical fiber. They used a sector instrument in addition to
an ion trap as an analyzer. Neither Vertes nor Lennon
systematically compared both irradiation geometries, nor
did they investigate the respective desorption mechanisms.
Ehring et aL5 used the transmission geometry to identify
potential thermal contributions to the MALDI process. In
most of their experiments, Ehring et al. prepared the sample
on a 200 nm gold layer which was sputtered onto the front
face of a substrate. The quartz substrate was then illuminated by the laser beam from behind so that light hit the
optically non-transparent gold layer rather than the sample
itself. These authors also mention however, that they
obtained spectra by transmission illumination without the
gold layer, i.e. with the sample prepared directly onto the
quartz substrate, similar to the procedure described in this
publication.
In the present paper, experiments comparing UV-MALDI
of peptides and proteins in transmission geometry and top
illumination are reported. Distinct differences in the respective plume dynamics are discussed.

EXPERIMENTAL
The experimentswere carried out with a time-of-flight mass
spectrometer (TOFMS)equipped with a linear port and a
single-stage ion reflector, built in the authors laboratory. A
nitrogen laser (A=337 nrn, 7=3 ns, Laser Science hc,
Newton, MA, USA) was used for desorption in both
irradiation geometries. The optical setup is shown schematically in Fig. 1.
In both top and transmission illumination, optical fibers
are employed to generate a homogeneous flat-top fluence
profile on the sample surface. For the top illumination, 2 m
of a silica/silica optical fiber (FG-200-LAT, 3M Company,
Received 3 September 1996
Accepted (revised) I5 October 1996

1874

MALDI IN TRANSMISSION GEOMETRY

attenuator

:%?!
substrate

fiber coupling units

metal support with stii

movable perpendicular to the plane

llluminrtlon
Figure 1. Optical layout for top and transmission illumination of MALDI samples.

West Haven, CT, USA) is used with a core diameter is

200 pm and a numerical aperture of 0.18. Due to multiple
reflections inside the fiber, the primary (Gaussian) laser
beam profile is converted into a flat-topped intensity profile
at the end face of the fiber, which is then imagined 1 :1 onto
the sample by two identical planoconvex lenses
(f=l2Omm) in infinite conjugation. The fiber optical
system has been described in detail previously.6
For the transmission illumination, another optical fiber of
141 pm core diameter and 0.22 numerical aperture is used
(Optran W 141/150A, Ceram Optec, Bonn, Germany). In
this case the output end of the fiber is placed directly behind
the rear side of a microscopic cover glass of 200 pm
thickness, while the sample is prepared on its front side. The
diameter of the irradiated area on the sample was measured
as 200pm, equal to that for top illumination. The beam
profile is nearly flat-topped, but, because of the free
propagation of the beam from the fiber exit face to the
sample, its edges are slightly less steep than the ones of the
top illumination. In order to ensure comparability of results
from the two irradiation geometries, the two fibers are
adjusted such that the identical spot is irradiated on the
sample.
A variable dielectric attenuator is used to adjust the laser
pulse energy. The energy of every laser exposure is
monitored by diverting part of the beam to fall on a
pyroelectric detector, which, in turn, is calibrated against an
accurate commercial energy meter (Laser Precision COT,
Yorkville, USA).
The unit for coupling the laser light into the top
illumination fiber is mounted on a translation stage and can
be moved into and out of the laser beam. In this way, it is
possible to change from top to transmission illumination
within a few seconds.
The cover glass carrying the sample is glued to a metal
support with a slit opening of approximately 0.8 x 10 mm
for the fiber to pass through. Because of constraints in the
instrument used, the sample could be moved over the fiber
tip along the slit in one dimension only. 2-D scan
arrangements will be realized in future experiments.
Desorbed ions are accelerated to an energy of 5 keV
applied by a potential between the metal support carrying
the sample cover glass at 5 kV and a three element
immersion ion lens at OV. The slight distortions of the
electric field caused by the slit under the sample were shown
by SIMION modeling not to negatively affect the mass

spectra. Charging effects of the dielectric substrate are also

not observed, provided the laser repetition rate stays at only
1 Hz or less. Ions are detected by a venetian-blind
secondary electron multiplier equipped with a conversion
dynode for post-acceleration of ions (5-20 kV, depending
on the mass of the substance investigated). A 300MHz
transient recorder (LeCroy 94504 Chesmut Ridge, NY,
USA) is used for data acquisition. The mass spectra are
transferred to a PC for further signal processing. The
instrument is equipped with a charge-coupled diode (CCD)camera in top view for sample observation and laser spot
localization.
2J-Dihydroxybenzoic acid (DHB) and cvHCCA were
used as matrices. Preparation of aHCCA was carried out
according to the fast evaporation method described by Vorm
et a1.*s9 using a solution of the matrix in acetone. In the
course of the experiments, it was found that a considerably
higher number of spectra could be obtained from the same
sample spot in transmission geometry if the evaporation was
slowed down somewhat by precooling the matrix solution to
ca. 4 C. Figure 2 shows electron micrographs of the two
preparations for comparison.
DHB/analyte samples were prepared by applying 2.5 pL
of matrix (10 g/L) plus 0.5 pL of analyte solution (0.1 g/L)
dissolved 1 :2 (v/v) in acetonitrile and a 0.1% aqueous
solution of trifluoroacetic acid. For proteins of higher mass,
2-hydroxy-5-methoxybenzoicacid (10 g/L, same solvent)
was added to the DHB solution in a volume ratio of 1 :9
(DHBs) for better performance. The cold air stream was
adjusted during the evaporation process to generate zones of
crystals comparably large in size (50-300 pm), surrounded
by microcrystalline areas. All chemicals were used as
purchased from commercial suppliers.

RESULTS
a-Cyano-4-hydroxycinnamicacid
Transmission MALDI from a thin layer preparation yields
peptide mass spectra which are equivalent to those obtained
from the sample spot under top illumination with respect to
peak pattern, signal intensities and mass resolution. As an
example, spectra of angiotensin I (Ang I, 1.3 kDa) obtained
in both irradiation modes are shown in Fig. 3. They show
almost exclusively protonated analyte molecules [M+ H]+
with a mass resolution of m/Am=2800. The matrix region

MALDI IN TRANSMISSION GEOMETRY

1875

50

IV\

1. I ;

125

500

mi z

ldoo

Figure 4. Single shot reflectron mass spectra of Angiotensin I (matrix:

DHB). (a) Transmission mode (H=450J/m2) (b) top illumination mode
(H=265Jim'). Both spectra were taken from the same sample.

Figure 2. Scanning electron micrographs of a-HCCAlacetone preparations. (a) Matrix solution at room temperature (b) Matrix solution at 4 C.

is dominated by [aHCCA+H]+ ions at mlz190 and

[aHCCA - OH]+ ions at mlz 172.
If prepared from a solution at room temperature, aHCCA
samples yield less than three spectra from the same area in
transmission mode and up to 30 in top illumination mode.
Precooling the matrix solution before preparation increases
the number of spectra which can be obtained in either mode
to over 100, while their quality is not affected in any way.

Dihydroxybenzoic acids
Standard DHB or DHBs preparations likewise allow one to
obtain transmission mode protein spectra which compare
well with those obtained in the top illumination mode. With

500
mlz

loo0

Figure 3. Single shot reflectron mass spectra of Angiotensin I (matrix:

aHCCA). (a) Transmission mode (H= 160 J/m) (b) top illumination mode
(H=174 J/m*). Both spectra were taken from the same spot.

this matrix, larger crystals, as typically found near the rim of

the preparation, serve as the best desorption sites. A large
number of different peptides and proteins in the mass range
from 1 to 150 kDa have been tested. Single-shot spectra of
Ang I (1.3 kDa) and bovine serum albumin (BSA, 66 m a ) ,
obtained from the same sample spots in both irradiation
geometries are shown in Figs 4 and 5 as examples. The
Ang I-peak in Fig. 4 is well resolved isotopically in both
spectra, similar to the spectra obtained with the (rHCCA
matrix. The two BSA spectra in Fig. 5 , obtained from DHBs
crystals are also virtually indistinguishable including the
relative abundance of multiply charged ions W+nH]+and
oligomeric [nM+ HI ions.
In all cases investigated, the mass resolution was equal
for both irradiation geometries. Up to a protein mass of
12 m a , mass resolution was sufficient to prove (by internal
calibration that protonated analyte molecules were preferentially formed in transmission as well as in top illumination.
From sampleareas of larger crystals far more than 100
transmission-mode mass spectra of consistent quality were
obtained from the same spot, notwithstanding the clear
evidence that in transmission mode almost all crystals
within the irradiated area are removed from the glass
substrate by the first few shots, leaving the irradiated area
virtually cleared of sample material (Fig. 6(a)). Only
crystals adhering to the substrate outside the desorption area
+

loo00

50000
mlz

15oooE

Figure5 Single shot reflectron mass spectra of BSA (matrix: DHBs).

(a) Transmission mode (H=380Jim') (b) top illumination mode
(H=350 J/m). Both spectra were taken from the same sample.

1876

MALDI IN TRANSMISSION GEOMETRY

and protruding into the irradiation area remain (Fig. 6(b)).

Inside the cleared areas only particles smaller than 1 pm in
size are found and lack any crystalline appearance. Their
shape suggests that they are recondensed ablation products
(Fig 6(c)).
The origin of ions recorded in later exposures, when the
desorption spot was already essentially cleared of sample
material, was determined in a separate experiment.A spot of
ca. 300 pm diameter was first cleared of material with 10
exposures by retracting the laser fiber by ca. 1 mm. Good
spectra were obtained even for the last of the 10 shots. The
fiber was then advanced again towards the substrate such

that only the center ca. 200 pm spot was irradiated. No ions
could be obtained from this center part of the original
ablation spot even at substantially higher fluences. The ions
seen in the mass spectra, therefore, must predominantly
originate from the rim crystals. A minor contribution from
the recondensed ablation products seems rather unlikely but
cannot be excluded completely. Considering this situation,
as illustrated in the Figs, it is certainly a surprise that in
transmission mode the absolute signal intensity is typically
only lower by a factor of 1.5 to 3 as compared to that for top
illumination, all other settings being comparable.

Fluence
For aHCCA-preparations, the threshold fluence (50 J/m2)
as well as the fluence used for obtaining the spectra of Fig.
3 (170 J/m2) were identical in top and transmission
illumination. Threshold fluence for spectra with a signal-tonoise ratio of 2 from DHB and DHBs preparations
(100 J/m2) were also equal for both illumination geometries. These observationshold even for later shots on a given
sample area, when most of the material has already been
removed from the spot in transmission mode. However, to
obtain DHB spectra of the intensity and quality of those
shown in Figs 4 and 5, the fluence in transmission geometry
had to be set, on average, as twice the value for desorption
for transmission as compared to top illumination (600 vs.
300 J/m2). Presumably this is related to the fact that the
local laser fluence at the rim of the desorption area is
certainly somewhat lower than in its center (see above).
Precision and mass accuracy
The precision (reproducibility) of the measured mass was
calculated for protonated "C-isotope of angiotensin I
(1296.7 Da) averaged over 20-30 single shot spectra
recorded from the same sample spot. Precision was
Am/m=5x
independent of the irradiation geometry
and the matrix (aHCCA or DHB). Mass accuracy was
determined as 80ppm for all these cases if matrix peaks
were used for the calibration of the mass scale. This value
is an upper limit, determined by the time resolution of the
transient oscilloscope.

Figure 6. Desorption areas of a DHB/Ang I-sample after 30 exposures

with H=630 Jim'. (a) Confocal-laser-micrograph of two irradiated spots,
(b) SEM micrograph of a single crystal at the rim of the desorption area,
(c) SEM micrograph of recondensed ablation products in the center of the
desorption area.

Location of ion emission in transmission mode

Experiments were conducted to investigate whether ions
from DHB crystals in transmission geometry are desorbed
from the top or the bottom surface. Large DHB single
crystals (typically 1 x 0.7 x 0.5 mm3length x width x height),
doped with cytochrome c, were fixed to the cover glass at
both ends with two strips of double-sided adhesive and
conducting tape, leaving a gap of ca. 200 pm thickness
between the bottom side of the crystal and the top face of
the cover glass in the center of the crystal. A light
micrograph of a mounted (and irradiated from below)
crystal is shown in Fig. 7. The penetration depth of the
337 nm-radiation into DHB is about 100 nm, much less than
the thickness of the single crystals (which, incidentally, is
also true for the crystals in a standard DHB preparation).
Mass spectra with a mass resolution only slightly less
than that for standard DHB preprations can be obtained
from these single crystals in top illumination. Spectra of the
same quality can likewise be obtained in transmission
geometry, remarkably not only from places near the edges
but also from desorption sites in the center of the rear
surface (see Fig. 7). After a number of exposures of the
bottom face of the crystal, specks of precipitated material

MALDI IN TRANSMISSION GEOMETRY

I877

material'' or metal" underneath the sample was irradiated,

however, so that no light could reach the sample directly.

Ion velocity

Figure 7. Photograph of a large cytochrome clDHB-single crystal (top

view). A desorption crater (several hundred shots!) on the rear side is
visible from the top side (arrow).

are clearly seen on the glass surface beneath the irradiated

spots. They represent ablation products of the sample.
In a further experiment a marker pen was used to cover
the top surface of a crystal with black dye. First the dye-free
rear side was irradiated in transmission geometry. The
resulting mass spectrum (Fig. 8(a)) shows almost exclusively matrix and analyte signals. After irradiation, no
damage to the dye coat was visible by optical microscopy.
Spectra taken in top illumination from the same crystal (Fig.
8(b)) are dominated by intense dye peaks, whereas the
cytochrome c signal intensity is much weaker than in the
transmission spectrum. In a complementary experiment, the
bottom side of a single crystal was covered with dye. In that
case the transmission spectrum showed strong dye signals
which were missing completely in the top illumination
spectrum.
These results, as well as the appearance of ablated
material underneath the crystals, clearly show that the
signals seen in transmission geometry essentially originate
from the bottom face of the crystals. They also eliminate the
possibility that shock-wave induced desorption from the top
face as was described by Lindner ef a/." and GrigorovI2for
direct laser desorption in transmission geometry has to be
considered. In those experiments, a suitable layer of organic

-."

Experiments with the cytochrome c (CC)/DHB-single

crystals described above were also performed using delayed
ion extraction to determine the mean initial axial velocity,
v ~of, the ions for the two irradiation geometries. In Fig, 9
the time-of-flight T of the CC ions is plotted against the
delay time T for ions registered in (a) linear-TOF and (b)
reflectron TOF modes of the instrument. It is immediately
evident that the transmission results differ significantly from
the top illumination data.
The mean initial velocity of the ions can be determined
from fits of the measured data to the equation for the timeof-flight T(T)(Eqn (1)). This was done under the following
assumptions: (1) upon desorption ions have a mean forward
initial velocity vB for top illumination and a backward mean
initial velocity of equal magnitude in transmission geometry. (2) There is sufficient space between the crystal rear
surface and the glass substrate for all the ions to be turned
around by the extraction field without major interaction with
the back-reflected neutral plume constituents under prompt
extraction. For delayed extraction the ion initial velocities
can be significantly modified through a plume reflection at
the substrate surface, provided the extraction delay time
exceeds a value of a few hundred nanoseconds. (3) Even
during the extraction delay a small, fringing, backwardpointing electric field Ef exists at the sample position.
Although this field is very weak (Ef=54V/mm),for delayed
extraction it has considerable influence on the overall flight

300-

285-

trammission-

t 285-

DHB DYE

W"
470

+0:O

0:s

l:O

1:s

2.0

2:s

$0

3,5

T I P

25.

0
0

illumination

1000

5Ooo

lo000

m/z
Figure 8. Mass spectra obtained from a cytochrome clDHB-single crystal
(top surface covered with dye). (a) Transmission mode (sum of 15 spectra)
(b) top illumination mode (sum of 9 spectra).

Figure9. Flight time versus ion extraction delay time of cytochrome c

ions desorbed from cytochrome c/DHB-single crystals in top and
transmission illumination. (a) linear TOF, (b) reflectron TOE For
explanation see text.
Top illumination: best tit:
v ( ~ ) = 6 6 0mls
(solid curves):
v(7)=(660+ 100) mls (dotted)
Transmission illumination: best f i t : Iv( 7)1=0 m/s. Iv,,,l%O (solid curves);
lv(~)l=O m/s. lv,,,l=O (dashed curve).

MALDI IN TRANSMISSIONGEOMETRY

1878

time and the initial ion velocity derived from a match of the
experimental results to Eqn (1).
-I

+/ - : transmission/top illumination

4 4
v(r)=vfi+7
m

where the symbols used have the following meanings:

m,q , ion mass and charge; D, length of acceleration region;
L, length of the field-free drift path; ER: electric field
strength in the ion reflector; 8,:angle between axes of drift
tube and ion reflector; E,: mean fringing field strength of ion
lens in the extraction region; z,,: axial starting position of the
ions; z( 7):position of ions when extraction field is switched
velocity of ions
on; v,: mean initial velocity of ions; ~(7):
when extraction field is switched on; U(7):potential at z( 7).
The parameters z, v, Ef are the components of the vectors
pointing along the axis of the mass spectrometer. They are
entered in Eqn (1) with a positive value if the direction
points from the sample towards the mass analyzer. The zaxis is the ion optical axis. It originates at the sample
support electrode which is connected to the full acceleration
potential. In the case of the linear TOE a zero length of the
ion reflector is assumed, resulting nominally in an infinite
ERand a vanishing last term of Eqn (1).
Some of the parameters in Eqn (1) are known (m,q),
others are accessible to direct measurement (L, D,ER, eR).
The ion starting position, Q , depends on the crystal used
and can be estimated fairly accurately. The potential U(z)
along the ion optical axis as well as the stray field of the lens
were calculated using SIMION7 software, taking into
account the influence of the dielectrics.
For the top illumination, the data obtained in reflectron as
well as in linear TOF mode give a best fit to Eqn (1)
assuming a forward mean initial velocity of (660-c50)m/s.
This value lies within the range of velocity data published
by other author^.'^-'^ In transmission mode, the data of the
linear as well as of the reflectron mode and for delay times
exceeding cu. 0.5 p,s are best fitted to Eqn (1) by assuming
a zero mean initial velocity v(7) at the time the extraction
field is switched on. Assuming, however, that, because of
the inverted irradiation geometry, the initial velocity vzo
would be zero as well and that desorbed ions would be
accelerated by the stray field, the resulting flight times
predicted by Eq. (1) would be represented by the dashed
curves in Fig. 9. These curves are not good fits of the
experimentally determined flight times. The contradiction
can, however, be resolved by assuming that the ions and
neutrals are desorbed with a substantial velocity towards the
glass substrate and collide with it, essentially unaffected by

the weak stray field before the strong extraction field is

switched on. The reflection of the plume at the substrate
could then lead to a randomization of the directions of the
initial velocities and, possibly to a partial or full thermalization of the plume through collisions of particles in parts of
the plume traveling in opposite directions. It is this
randomization which results in the zero mean velocity of the
ions at the time the extraction field is switched on. These
assumptions are further supported by the flight times
measured for prompt extraction. As predicted by the model
presented above, the strong prompt-extraction field will turn
around the ions before they can collide with the reflected
neutral plume constituents. The measured flight times for
prompt extraction are, therefore, extended by the turnaround-time compared to those for delay times of, for
example 0.5 ~ sA .velocity component parallel to the crystal
surface either from the desorption or through the collisions
in the plume must, of course, also be assumed, to allow the
ions to escape from underneath the crystal after the
extraction field is switched on.
The data obtained for standard preparations are not as
conclusive as those for single crystals. For a standard DHB
preparation, a best fit was obtained for a mean forward
velocity of 550-600m/s for top illumination. In the
transmission mode, a velocity of 350-550 m/s gave the best
fit with a considerable variation from preparation to
preparation and from spot to spot. This indicates only partial
randomization, which is not too surprising, considering the
fact that ions are, in all likelihood, mostly generated from
the rim crystals protruding into the desorption spot with
faces inclined considerably with respect to the substrate
surface (see Fig. 6(b)). The (uHCCA matrix behaved
differently again. In top illumination the mean forward
velocity was estimated to be ca. 350m/s. The lower
velocity than that obtained for DBH is in good agreement
with observations by Juhasz et all9In transmission the bestfit mean velocity was only 90 m/s.

THE THERMALIZED PLUME MODEL

In the following, the model of a plume thermalization under
the special constraints of transmission desorption from large
single crystals will be discussed in more detail.
As shown, desorption is essentially taking place from the
rear surface of the crystal in transmission geometry. It is,
probably, safe to assume that the initial phase of the plume
expansion is identical in the transmission- and in the topillumination modes, because free surfaces of the same
sample are irradiated in both cases. It is well documented in
the literaturezGz2that the plume expands with a strong
velocity component of cu. 600m/s normal to the crystal
surface. The dynamic behavior is certainly determined by
the neutral DHB matrix molecules being present in high
molar excess.6 Within experimental error the determined
velocities of the cytochrome c ions are about the same as
those of desorbed neutral matrix molecules.23Taking these
values and a typical distance from the crystal rear surface to
the substrate of 200 p,m, the plume hits the substrate at a
time of ca. 300 ns after exposure, i.e. before the extraction
field is switched on. Upon collision with the substrate at
least a fraction of the molecules and ions in the plume is
assumed to be back-reflected, predominantly elastically, and
the back-travelling front part of the plume will collide head
on and mix with the still forward traveling rear part. The
many collisions during this mixing process are assumed to
be the main cause of the randomization of the directions of

MALDI IN TRANSMISSION GEOMETRY

the initial velocity. Two extreme scenarios are conceivable

for the ensuing plume processes. The details of the
collisions of the plume with the substrate under the given
conditions are not known. For simplicity of argument, the
energy loss to the substrate through (partially) inelastic
collisions is, therefore, assumed to be negligible in both
cases. Also, the collisions of neutrals and ions with the
substrate or crystal surface after partial or full thermalization are not frequent enough during the few ps of delay time
to result in temperature equilibration between the substrate
and the plume. In the one extreme case, all collisions among
neutrals and ions in the plume are assumed to be fully
elastic. In this case, all the kinetic energy originally
associated with the centre-of-mass velocity of the plume
will be randomized among the six external degrees of
freedom (3 translational, 3 rotational) of all particles.
Taking into account the large molar excess of matrix
molecules, carrying the overwhelming fraction of the initial
kinetic energy in the expanding plume, their average kinetic
energy of translation will decrease by a factor of 2 and their
average speed by a factor of @ through the collisions. For
a full thermalization of the external degrees of freedom of
all particles in the plume their root mean square (RMS)
speed can be related to a translational temperatureby

v,,=

where k is Boltzmanns constant.

For this model the translational temperature of the
thermalized plume would be ca. 1300 K.In contrast to the
desorption plume before thermalization, the RMS speed of
the plume constituents will decrease with the square root of
the mass, giving an RMS-speed of only 18 m/s for a protein
of mass 100 kDa, or of 184 m/s for a 1 kDa peptide and
466 m/s for matrix ions of mass 155 Da. Also, for this
model, the temperature, associated with the internal degrees
of freedom of the ions, would remain unchanged and no
difference in fragmentation would be expected.
If, in the other extreme, the collisions among the plume
constituents are assumed to be inelastic, the collisions will
result in a randomization of the total energy among all
accessible internal and external degrees of freedom. It is
reasonable to assume that matrix as well as analyte
molecules/ions carry an excitation of their internal degrees
of freedom from the desorption process, which corresponds
to a temperature at most somewhat above that of thermal
decomposition in equilibrium. This can be concluded from
the observed metastable decay of MALDI ions, typically on
the micro- to millisecond time scale. Compared to the total
internal temperature of then, at most, a few hundred
degrees, the centre-of-mass kinetic energy of the plume
constituents will be negligible. In this case, the equilibrium
temperature of the ions internally and with respect to their
translational motion would be, at most, cu. 500 K. Again no
difference in ion fragmentation would be expected in this
case and neither is it observed. In reality, only a partial
thermalization and a situation somewhere in between the
two extremes discussed will, most probably, be attained.
Another, though less likely, model could be based on the
idea that the ions become redirected in the space between
the crystal and the substrate and are ejected from this space
in a (laminar) flow, of high speed, parallel to the substrate
surface.

1879

DISCUSSION
The experimental results obtained for the gap-mounted
single DHB crystals constitute convincing evidence that the
desorption plume thermalizes, if confined in a suitably
narrow space and if given a time of cu. 0.5 IJ.S or more. The
results for the standard preparations are not quite as
conclusive. For DHB preparations it is reasonable to assume
that the desorption takes place mainly from crystal surfaces,
inclined relative to the substrate surface as evidence by Fig.
6(b). This wiIl diminish the velocity component along the
ion optical axis and will result in plumes hitting the
substrate surface at a non-normal angle. The mixing of the
counter-travelling parts of the plume will then be less
perfect, leading to a less efficient thermalization. This lack
of efficient thermalization can explain why the measured
mass resolution under prompt extraction is equal for top and
transmission illumination (see Figs 3-5). No such morphological detail has as yet been acquired for the (uHCCA
preparation and no explanation can be given for the
observed velocity decrease in transmission versus top
illumination geometry.
The experimental results and the model suggest, on the
other hand, that suitable ion source geometries, possibly in
conjunction with delayed ion extraction, should allow for an
optimization of the mass resolution as a consequence of
thermalization. Two main independent features of a thermalfzed desorption plume are expected to contribute to the
attainable mass resolution. First, ions travelling in opposite
directions along the ion optical axis will have a time
dispersion equal to the turn-around-time in the extraction
field. The turn-around-time will become minimal for strong
extraction fields. Secondly, the plume will expand in the
axial direction in particular if extraction is delayed. Ions at
different axial positions will be accelerated through different potentials upon extraction and will, therefore, have an
energy dispersion. In contrast to the turn-around-time
diipersion, the energy dispersion becomes minimal for a
low extraction field strength. A narrow, confined space for
the thermalization may also help, not only in an efficient
thermalization, but also in keeping the plume confined to a
short axial extent. The energy dispersion can, to a certain
extent, be compensated for by an ion reflector in the TOF
analyzer. In some ion source geometries such as the one
realized for the single crystals, ions may also leak out from
the confining thermalization space for some time, which
will also introduce a time dispersion. The first two
independent influences on the mass resolution can be
combined into a single equation. Thus, including the
possibility for a split ion extraction with an electric field
strength E,,,, in the extraction region followed by a second
region where the ions are accelerated to a final energy qU,
the mass resolution can be calculated to be:

provided L %=D
where: Az is the axial extent of the plume upon ion
extraction, Lq the equivalent length of ion path; T the plume
temperature; Eexm the electric field strength in the ion
extraction region, and U the total ion acceleration potential.
The first term of Eq. (3) describes the influence of the axial

M M D I IN TRANSMISSIONGEOMETRY

1880

plume extent, the second the turn-around-time. It is worth

noting that both terms predict a mass resolution independent
of the ion mass in contrast to the observed decrease of mass
resolution with increasing mass in standard MALDI-TOF
instruments.
Obviously different optimization strategies are needed for
reflectron and linear TOF instruments. For reflectron
instruments the energy dispersion, represented by the first
term of Eqn (3), will be compensated by the ion reflector.
The highest mass resolution will then be achieved with a
single-stage ion extraction, with U and Lq as large as
possible and D and T as small as possible.
For a linear TOF instrument mass resolution is maximized if the two terms of Eqn (3) are set equal. In this case
the extraction field strength is given by:

L XAz

(4)

and the best mass resolution will be:

MALDI-MS. Model calculations do indeed show that a

significant improvement in mass resolution can be expected
from a thermalized plume, if conditions are optimized for
the transmission geometry.
The major aim for future work, therefore, will be the
development of suitable source geometries, which will
allow thermalization in a very small volume (Az= 10 pm).
In combination with a suitable extraction field geometry,
possibly including delayed-ion extraction, the potential for
the improvement of the mass resolution should manifest
itself particularly in the high-mass range. In this respect it is
conceivable that the thermalized plume could allow the use
of relatively low acceleration potentials and also be an
alternative to orthogonal extraction sources,24because both
techniques are based on the elimination of the high axial
velocities. As another application, one could think of the
coupling a thermalized MALDI source to a FTICR mass
spectrometer, for which the high initial energies of MALDI
ions cause severe trapping problems.

Acknowledgment
This work was done in partial fulfillment of the requirement for the Ph.D.
of Martin Schiirenberg at the University of Miinster.

For a good mass resolution, the extraction field strength

E,,, should be small and the total ion energy U as high as
possible. This is best achieved with a split ion extraction.
Overall, however, increasing the total ion energy U and the
field free drift length Lq, as well as decreasing Az and T,
will increase the mass resolution.
Equation (5) can be used to calculate the expected mass
resolution for the instrument used in the experiments. With
L=2.3m, D = 8 m m and U=5kV, and assuming
Az= 100 pm, a mass resolution of only 80 is calculated to
result. This is close to the actually observed value. Equation
(9,on the other hand, predicts that a much better mass
resolution should be achievable with an optimized split ion
extraction. Assuming a plume translational temperature of
1300K (see above) the optimal extraction field strength
would be ca. 64 V/mm with a mass resolution of 490. For
T=500 K, the field strength is 50 V /mm and the resolution
is 630.

CONCLUSION AND OUTLOOK

Using transmission illumination geometry, protein mass
spectra can be acquired from slightly modified DHB and
aHCCA standard preparations that are absolutely equivalent in quality to spectra obtained by (conventional) top
illumination. The transmission geometry therefore can
provide a real alternative to the latter, especially in the case
where either the brightness of the laser (diode laser!) or the
geometric restrictions in the ion source do not allow for an
irradiation of the top-surface of the sample.
Much more important, however, is the observation that,
under certain conditions, the mean velocity of the ions in the
plume becomes zero. This thermalized plume provides for
the first time a MALDI-source without a strongly forwardpeaked velocity distribution. The jet-like plume expansion
in conventional MALDI sources with its high mean velocity
and width of the distribution is presumably the most
important limiting factor for high mass resolution in

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