Determination of internal energy distributions of laser electrospray mass spectrometry using thermometer ions and other biomolecules

Article abstract of DOI:10.1007/s13361-014-0936-6

(Figure Presented) The internal energy distributions for dried and liquid samples that were vaporized with femtosecond duration laser pulses centered at 800 nm and postionized by electrospray ionization-mass spectrometry (LEMS) were measured and compared

Full text of DOI:10.1007/s13361-014-0936-6

B American Society for Mass Spectrometry, 2014
J. Am. Soc. Mass Spectrom. (2014) 25:1572Y1582
DOI: 10.1007/s13361-014-0936-6
RESEARCH ARTICLE
Determination of Internal Energy Distributions of Laser
Electrospray Mass Spectrometry using Thermometer Ions
and Other Biomolecules
Paul M. Flanigan IV,1,2 Fengjian Shi,1,2 Johnny J. Perez,1,2 Santosh Karki,1,2
Conrad Pfeiffer,1 Christian Schafmeister,1 Robert J. Levis1,2
1Department of Chemistry, Temple University, Philadelphia, PA 19122, USA
2Center for Advanced Photonics Research, Temple University, Philadelphia, PA 19122, USA
Abstract. The internal energy distributions for dried and liquid samples that were
vaporized with femtosecond duration laser pulses centered at 800 nm and
postionized by electrospray ionization-mass spectrometry (LEMS) were measured
and compared with conventional electrospray ionization mass spectrometry (ESI-
MS). The internal energies of the mass spectral techniques were determined by
plotting the ratio of the intact parent molecular features to all integrated ion
intensities of the fragments as a function of collisional energy using
benzylpyridinium salts and peptides. Measurements of dried p-substituted
benzylpyridinium salts using LEMS resulted in a greater extent of fragmentation
in addition to the benzyl cation. The mean relative internal energies, GEint9 were
determined to be 1.62 0.06, 2.0 0.5, and 1.6 0.3 eV for ESI-MS, dried LEMS, and liquid LEMS studies,
respectively. Two-photon resonances with the laser pulses likely caused lower survival yields in LEMS
analyses of dried samples but not liquid samples. In studies with larger biomolecules, LEMS analyses of
dried samples from glass showed a decrease in survival yield compared with conventional ESI-MS for
leucine enkephalin and bradykinin of ~15% and 11%, respectively. The survival yields for liquid LEMS
analyses were comparable to or better than ESI-MS for benzylpyridinium salts and large biomolecules.
Keywords: Femtosecond vaporization, Electrospray ionization, Ambient mass spectrometry, Internal energy
deposition
Received: 7 February 2014/Revised: 2 May 2014/Accepted: 16 May 2014/Published online: 11 July 2014
methods. The survival yield (SY) method is a common
approach for determining the internal energy of a mass
Introduction
he “soft” ionization methods, including electrospray
spectral technique [7]. This method assumes that all ions
with an internal energy below the critical energy, Eo, do not
dissociate whereas all ions with an internal energy above the
threshold do so. In theory, only the molecular ion is
observed when EintGEo and only fragment ions are observed
when Eint9Eo. The survival yield measures the fraction of
ions with an internal energy below the critical energy and
can be calculated using Equation (1),
T
ionization (ESI) [1, 2] and matrix-assisted laser desorp-
tion/ionization (MALDI) [3–5], enable the detection of intact
macromolecular ions with minimal fragmentation. By
comparison, electron impact ionization creates a distribution
of internal energies, Eint, for a given molecule with a higher
average because of the typical ~70 eV electron beam [6].
The internal energy of the ions controls the extent of
fragmentation in the obtained mass spectrum. Measurements
of the internal energy distribution deposited by a mass
spectral method are important for developing improved
Z
Eo
IðMþÞ
h
i
SY ¼
¼
PðEÞdE
ð1Þ
X
IðMþÞ þ
IðFþÞ
0
Electronic supplementary material The online version of this article
(doi:10.1007/s13361-014-0936-6) contains supplementary material, which
is available to authorized users.
where I(M+) and I(F+) are the intensity of the molecular and
fragment ions observed in the mass spectra, respectively.
The internal energy distribution, P(E), can be determined by
Correspondence to: Robert J. Levis; e-mail: rjlevis@temple.edu

P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
1573
taking the derivative of the survival yield as a function of
energy using a set of ions having the same internal energy
distribution but with different critical energies. The experi-
mental internal energy distributions are collective values
resulting from the various energetic states of the ionized
analyte that evolve from the beginning of the analysis to the
mass spectral detection. A set of ions for which the critical
energy values are well-characterized and vary over a suitable
range, called “thermometer” molecules, are used to deter-
mine the internal energy [7, 8].
analysis. On the other hand, the folded state of lysozyme
was better preserved using liquid LEMS analyses than
conventional ESI-MS when subjected to high collision
induced dissociation (CID) energies [31].
Here, we use the survival yield method to characterize the
internal energy deposited during LEMS analyses of dried
and liquid samples and make comparisons to conventional
electrospray-mass spectrometry analyses. Para-substituted
benzylpyridinium ions are used to investigate the internal
energy deposition of small molecules and the peptides
leucine enkephalin and bradykinin are used to gain insight
on the energy deposited into larger molecules.
The most common thermometer ions used are a set of
substituted benzylpyridinium salts because of the relatively
simple fragmentation pattern resulting from the loss of
neutral pyridine leaving the benzyl cation for mass analysis,
which is shown in Scheme 1. These molecules have similar
masses, structures, and degrees of freedom, leading to well-
char acteri zed internal energy distributions.
Benzylpyridinium ions have been extensively used to
determine the “softness” of a given mass spectral technique
and to determine how various parameters affect the internal
energy deposition for fast atom bombardment (FAB) [9],
secondary ion mass spectrometry (SIMS) [10, 11], MALDI
[12–14], atmospheric pressure MALDI (AP-MALDI) [12,
15], ESI [8, 16–20], desorption electrospray ionization
(DESI) [21, 22], surface-assisted laser desorption/ionization
(SALDI) [23], silicon nanoparticle-assisted laser desorption/
ionization (SPALDI) [24], direct analysis in real time
(DART) [25], surface acoustic wave nebulization (SAWN)
[26], and laser ablation electrospray ionization (LAESI) [27].
A recently developed ambient mass spectrometric tech-
nique, laser electrospray mass spectrometry (LEMS), em-
ploys an intense 800 nm femtosecond laser to induce
nonresonant vaporization of samples from a surface,
followed by capture and postionization by an electrospray
ionization source. No sample preparation is required in the
LEMS method because of nonresonant, multiphoton vapor-
ization from the surface by intense femtosecond laser pulses
(1013 W/cm2). This method has been used for mass spectral
analysis of small biomolecules [28, 29], proteins [30–32],
lipids [33], explosives [34–36], smokeless powders [37],
narcotics [38], pharmaceuticals [38], and tissues [39, 40].
Although the internal energy distribution after femtosecond
laser vaporization has not been measured, previous studies
have shown differences in the amount of fragmentation
when comparing mass spectra obtained with LEMS and
conventional electrospray mass spectrometry. LEMS analy-
ses of dried irinotecan HCl [29] and dried
tetrabutylammonium iodide [41] have resulted in more
fragmentation compared with conventional electrospray
Experimental
Synthesis and Preparation of p-Benzylpyridinium
Salts
All starting materials for the synthesis of p-
benzylpyridinium (BzPy) salts were used as received from
Sigma Aldrich (St. Louis, MO, USA). The benzylpyridinium
salts were synthesized as described [24, 26], with the
reactions performed under an argon atmosphere. The
methoxy- (p-MeO), methyl- (p-Me), cyano- (p-CN), and
nitro- (p-NO2) p-substituted benzylpyridinium salts were
synthesized by mixing the respective p-benzyl chloride with
an excess of anhydrous pyridine in dry acetonitrile at room
temperature overnight. The p-chlorobenzylpyridinium (p-Cl)
salt was prepared by heating a solution of the benzyl halide
and excess pyridine in acetonitrile to 80°C for 3 h. After
completion of the reaction, the salts were precipitated with
diethyl ether that was subsequently removed by evaporation,
diluted with 1:1 (v:v) acetonitrile/water, frozen with liquid
nitrogen, and lyophilized. The purified salts were diluted to
2 mM stock solutions using HPLC grade water and stored in
the refrigerator prior to analysis. After initial experimenta-
tion and for verification of purity, the benzylpyridinium salts
were concentrated in DMSO, purified using an Xterra Prep
C18 (10×150 mm, 5 μm particle) column (Waters Corpo-
ration, Milford, MA, USA) on an Agilent 1100 series HPLC
(Agilent Technologies, Inc., Santa Clara, CA, USA),
lyophilized, and diluted in methanol/water using HPLC
grade methanol (Fisher Scientific, Fair Lawn, NJ, USA).
The stock BzPy solutions were diluted to 1 mM in 1:1
(v:v) methanol/water and 20 μM in 1:1 methanol/water with
1% acetic acid for LEMS analyses of dried samples and
conventional electrospray analyses, respectively. Solutions
for LEMS analyses from liquid samples were prepared as
follows: 500 μM in 1:1 methanol/water for p-MeO, p-Me,
and p-Cl BzPy ions; 1.71 mM in water for p-CN BzPy; and
1 mM in 1:1 methanol/water for p-NO2 BzPy.
Other Materials and Sample Preparation
Scheme 1. Dissociation pattern of p-substituted
benzylpyridinium ions into the benzylium cation and neutral
pyridine
All solid samples were used without further purification and
were diluted to obtain stock solutions of 1 mM. The peptide

1574
P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
samples of leucine enkephalin (Tyr-Gly-Gly-Phe-Leu) ace-
tate salt hydrate (Sigma Aldrich) and bradykinin (Arg-Pro-
Pro-Gly-Phe-Ser-Pro-Phe-Arg) (AnaSpec, Fremont, CA,
USA) were diluted in water, whereas the pharmaceutical
irinotecan HCl (Sigma Aldrich) was diluted in 1:1 (v:v)
methanol/water. For individual conventional electrospray
analyses, the peptides and irinotecan HCl were diluted in 1:1
methanol/water with 1% acetic acid to obtain final concen-
trations of 25 μM and 20 uM, respectively. For LEMS
analyses of the peptides as dried and liquid samples, the
solutions were diluted to 500 μM in 1:1 methanol/water. The
stock solution was used in LEMS analysis of dried
irinotecan HCl.
Dried samples for LEMS analysis were prepared by
spotting 15 μL aliquots on glass microscope slides (Fisher
Scientific) that were cut to 7×7 mm, with each solution
spotted on multiple sample slides. After allowing the sample
slides to dry, the procedure was repeated once for irinotecan
HCl and twice for the benzylpyridinium salts and the
peptides, resulting in 30 and 45 μL of each solution
deposited onto multiple sample slides, respectively. Samples
for leucine enkephalin and irinotecan HCl were also spotted
on 7×7 mm stainless steel sample slides for comparison with
vaporization from the glass slides. To perform a LEMS
measurement on a liquid sample, 10 μL was spotted on a
2.2×3.8 cm stainless steel slide and analyzed, with at least
seven such samples prepared for every analyte. The sample
slides were placed into the LEMS source chamber on a
metal plate attached to a three-dimensional translational
stage, which allowed raster scanning in order for new
sample to be analyzed with every laser pulse. For temper-
ature-controlled experiments, the translation stage was
cooled using a Neslab RTE-100 refrigerated bath circulator
(Neslab Instruments, Inc., Newington, NH, USA) and a
custom brass plate.
varied from 0.7 to 2.1 mJ, corresponding to an intensity
range of ~1.4 to 4.2×1013 W/cm2.
After vaporization, the sample material is captured,
ionized, and transferred into a mass spectrometer by an
electrospray source (Analytica of Branford, Inc., Branford,
CT, USA). The acidified electrospray solvent, 1:1 (v:v)
methanol/water with 1% acetic acid, was pumped through
the ESI needle by a syringe pump (Harvard Apparatus,
Holliston, MA, USA) at a flow rate of 3 μL/min. The
electrospray plume was dried by countercurrent nitrogen gas
primarily at 180°C before entering the inlet capillary. For
drying gas temperature experiments, the temperature was
varied from 105 to 180°C. Fragmentation of the molecules
was performed by increasing the voltage between the
capillary exit and the skimmer of the hexapole in the
spectrometer, as seen in Figure 1. An ESI solvent back-
ground mass spectrum was acquired before vaporization of
each sample set to allow background subtraction of solvent-
related peaks. Each LEMS experiment resulted from the
averaging of 50 laser shots, which were averaged using a
digital oscilloscope (LeCroy Wavesurfer 422; LeCroy Co.,
Chestnut Ridge, NY, USA). At least 15 and seven separate
measurements were performed for LEMS analyses of dried
and liquid samples, respectively. Three mass spectra, each
consisting of averaging for 5 s, were collected for conven-
tional ESI-MS analyses.
Calculation of Survival Yields and Internal Energy
Distributions
The raw mass spectral data files obtained from the digital
oscilloscope were saved using a custom Labview 8.5
program (National Instruments, Austin, TX, USA) and
imported into the Cutter program [42] for integration of the
peaks of interest, which were confirmed by tandem MS
using conventional ESI-MS on a high resolution Bruker
MicrOTOF-Q II mass spectrometer (Bruker Daltonics,
Billerica, MA, USA). The M+ and M+-pyridine features for
the BzPy ions, the M+H+ and [M+2H+]2+ features for
leucine enkephalin and irinotecan HCl, and the [M+2H+]2+
and [M+3H+]3+ features for bradykinin were isolated and
fragmented using CID cell voltages ranging from 5 to 50 eV.
In the benzylpyridinium MS/MS studies, the in-source
collision induced dissociation (ISCID) voltage was adjusted
to enable significant intensity of the M+-pyridine feature for
isolation and further fragmentation. All of the fragments
verified by high resolution tandem MS were used in the
calculations of the survival yields, including those other than
the M+-pyridine ion for the thermometer analysis.
Laser Electrospray Mass Spectrometry
The instrumentation for laser vaporization, electrospray
postionization, and mass spectral detection has been previ-
ously described in detail [28, 30, 31, 33–35, 38–41]. A
Ti:sapphire oscillator (KM Laboratories, Inc., Boulder, CO,
USA) seeded a regenerative amplifier (Coherent, Inc., Santa
Clara, CA, USA) to create 70 fs pulses centered at 800 nm.
The laser pulse, which was focused to a spot size of
~300 μm in diameter using a 16.9 cm focal length lens with
an incident angle of 45° with respect to the sample, was
operated at 10 Hz to enable analysis of fresh sample and to
synchronize with the hexapole operating in the trapping
mode [28, 40]. The power of the laser was attenuated using a
quarter-wave plate and a polarizing beam-splitting cube
(CVI Laser Optics, Albuquerque, NM, USA) to a pulse
energy of 1.3 mJ for the majority of the experiments,
resulting in an intensity of ~2.6×1013 W/cm2 at the area
sampled. For laser energy studies, the pulse energy was
The method used to calculate the internal energy
distributions was based on the survival yield method [7].
The SY values calculated using Equation 1 were plotted as a
function of the capillary exit voltage to reveal breakdown
curves. For thermometer ions, the SY values for the five
BzPy ions at one capillary exit voltage (at 280 V) were
plotted as a function of their respective critical energies, Eo,

P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
1575
Figure 1. Schematic of the instrumental setup. The experimental region is where ESI and LEMS are peformed and the in-
source collision induced dissociation (ISCID) region is where fragmentation occurs in the mass spectrometer by varying the
capillary exit voltage
which were previously reported [8]. Two assumed SY
values of 0% and 100% at 0 and 3.5 eV, respectively, were
added to the SY curve to approximate low and high energy
dissociation thresholds where complete or no fragmentation
would be observed. OriginPro 9 (OriginLab Co., Northamp-
ton, MA USA) was used to fit the sigmoidal curves to the
experimental data points, differentiate the sigmoidal curves
for the internal energy distributions and calculate the mean
internal energies (GEint9). The sigmoidal curve was fit using
a Boltzmann function, as seen in Equation (2), where E is
the internal energy, Ec is the energy at the centroid of the
curve, A1 is the initial SY value, and A2 is the final SY
value.
mass spectra at low and high collision energies for the other
three thermometer ions, p-MeO, p-CN, and p-NO2 are
presented in Figure S1 in Supplementary Material. The m/z
values for all features of interest are denoted on the mass
spectra with the parent molecular (M+) and parent minus
A1 − A2
SY ¼
þ A2
ð2Þ
1 þ eðE − E Þ=dE
c
Safety Considerations
Appropriate laser eye protection was worn by all lab
personnel.
Results and Discussion
Mass Spectra of Thermometer Ions Using LEMS
To characterize the energy distributions of molecules
vaporized by nonresonant femtosecond laser pulses centered
at 800 nm, the mass spectra of five para-substituted
benzylpyridinium (BzPy) ions were measured using laser
electrospray mass spectrometry (LEMS) and compared with
conventional ESI-MS. LEMS measurements were collected
for BzPy ions from both dried and liquid states on glass and
metal substrates, respectively.
Figure 2 shows the mass spectra obtained at low and high
collision energies for p-Me and p-Cl BzPy ions analyzed
using conventional ESI-MS (Figure 2a and d), dried LEMS
(Figure 2b and e), and liquid LEMS (Figure 2c and f). The
Figure 2. Mass spectra obtained for (a)–(c) p-Me and (d)–(f)
p-Cl benzylpyridinium ions using (a) and (d) conventional
electrospray, (b) and (e) LEMS of dried samples from glass,
and (c) and (f) LEMS of liquid samples from metal. The plots
show the fragmentation of the ions at low and high capillary
exit voltages of 130 and 230 V as black and red lines,
respectively. The M+ and M+-pyridine features are denoted
by the blue squares and red circles, respectively

1576
P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
pyridine (M+-Pyr) features highlighted as blue squares and
red circles, respectively. The corresponding masses and
dissociation energies of the BzPy salts are shown in Table 1.
The intensities of the parent and fragment ions are related to
the internal energies resulting from each of the analysis
techniques. We note that peaks other than the M+ and M+-
Pyr features were observed in the mass spectra, specifically
in the LEMS analyses of dried thermometer ions. Some of
these features, such as those at m/z 78 for all BzPy ions,
were the most intense peaks in the LEMS analyses of dried
thermometer ions. Fragments other than M+-Pyr can also be
seen in the conventional ESI-MS and LEMS analyses of
liquid samples as well, but were less prevalent than in dried
LEMS analyses. Negative features observed in the blank-
subtracted mass spectra result from the vaporized analytes
competing for charge and thus altering the solvent ion
distribution. Positive features in the mass spectrum that are
not labeled in the blank-subtracted mass spectra are solvent-
related features.
To determine whether the mass spectral features other
than M+-pyridine, such as m/z 78, resulted from fragmenta-
tion of the BzPy ions, tandem MS was performed on the
isolated M+ and M+-pyridine ions in a collision cell using a
high resolution mass spectrometer with conventional ESI-
MS. The ESI-MS/MS mass spectra are shown in Figures S2
to S6 in Supplementary Material. Most of the fragment ions
detected in our ESI-MS and LEMS experiments have been
observed previously [26, 43]. Table S1 in Supplementary
Material lists the observed ions as well as those that were
included in the survival yield calculations for the LEMS
experiments. The additional fragments observed in LEMS
experiments result from reaction pathways other than the
simple loss of a neutral pyridine. This includes intramolec-
ular rearrangements [43] and rearrangement to the tropylium
cation [20, 44, 45].
Figure 3. Breakdown curves for the p-substituted
benzylpyridinium ions as a function of capillary exit voltage for
(a) conventional ESI-MS, (b) LEMS of dried samples from glass,
and (c) LEMS of liquid samples from metal. The insets in (b) and
(c) show the effect of the pulse energy on survival yield at a
capillary exit voltage of 230 V for p-Me and p-Cl BzPy dried and
liquid samples, respectively. The box around the symbols at a
capillary exit voltage of 280 V indicates the voltage chosen for
the internal energy distribution calculations
BzPy in the breakdown curves for ESI-MS (Figure 3a) are in
good agreement with the critical energy values, Eo. Ions with
high critical energy values (p-NO2 BzPy) do not dissociate
as easily as those with low Eo values (p-MeO), resulting in
less fragmentation and a larger yield of parent ions for the
high Eo molecules. p-MeO had the lowest survival yield
values in all experiments, in agreement with its low critical
energy.
Survival Yields and Internal Energy Distributions
for LEMS Compared with ESI-MS
Survival yields for the five thermometer ions were calculated
using Equation (1) and plotted as a function of collision
energy for ESI and LEMS experiments in Figure 3. The
descending order of survival yield for p-NO2 to p-MeO
The LEMS measurements differ in several respects from
the survival yield/critical energy trends observed in the ESI
measurements. Although p-CN and p-NO2 BzPy ions have
the highest critical energy values, they had lower survival
yields than p-Me and p-Cl in dried LEMS analyses
(Figure 3b) for capillary exit voltages ranging from 130 to
230 V. At 255 and 280 V, p-CN had the highest survival
yield but p-NO2 was still lower than p-Me and p-Cl. A
similar trend is noted for the analysis of liquid samples using
LEMS (Figure 3c), although the overall survival yields were
much higher in comparison with dried LEMS analyses. p-
Table 1. Dissociation Critical Energies and Major Mass Spectral Features
Expected for the Benzylpyridinium Ions
Analyte
Eo
M+
(m/z)
M+ - Pyridine
(m/z)
(eV)a
p-MeO
p-Me
p-Cl
p-CN
p-NO2
1.51
1.77
1.90
2.10
2.35
200.3
184.3
204.7
195.2
215.2
121.2
105.2
125.6
116.1
136.1
aTaken from Reference [8].
M+: molecular ion, Eo: critical energy.

P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
1577
NO2 BzPy had the second highest survival yield at 280 V in
the LEMS analysis as a liquid sample, although it should
have the highest SY. The non-intuitive increase in survival
yield with increasing capillary exit voltage for p-CN and p-
NO2 resulted from the combination of the masking of low
mass ions due to optimization for high mass ions and the
enhanced dissociation to low mass ions at higher collision
energy. The enhanced dissociation and masking effect is
supported by the normalized total ion yield plots (Figure S7
in Supplementary Material), which show a decrease in the
total ion yield as a function of capillary exit voltage for
analyses with ESI-MS and LEMS of dried samples.
Conversely, the total ion yields for most of the BzPy ions
vaporized as liquid samples increased or remained un-
changed, suggesting that less energy is deposited into the
internal modes of the liquid sample. The shift in the ion
transmittance was most apparent in the survival yields from
the vaporization of dried BzPy ions, presumably because the
interaction with the laser pulses resulted in a greater degree
of fragmentation. We propose that dissociation of the benzyl
cation fragments into a mass range not detected by our mass
spectrometer plays a major role in the survival yield
calculation.
Figure 4. Internal energy deposition of LEMS compared
with conventional ESI-MS. (a) Survival yield values were
plotted as a function of critical energy, Eo, at a capillary exit
of 280 V. The derivatives of the sigmoidal curves produced
the (b) internal energy distributions [P(E)]. The sigmoidal fit
R2, mean internal energy, GEint9, and full width at half
maximum (FWHM) internal energy values for each technique
are shown as tables in the figure. Note that the sigmoidal fit
for dried LEMS analyses (red) does not include the survival
yield for the p-NO2 benzylpyridinium ion
To calculate the internal energy distributions [8], the
survival yields obtained at a capillary exit of 280 V were
plotted as a function of critical energy, Eo, in Figure 4a. This
exit voltage displayed the greatest spread in the internal
energy distribution for ESI-MS. The fit of the sigmoidal
curve using the Boltzmann function (Equation (2)) had R2
values of 0.992, 0.997, and 0.951 for conventional ESI-MS,
dried LEMS, and liquid LEMS analyses, respectively. Note
that the survival yield for the p-NO2 benzylpyridinium ion
was not included in the sigmoidal fit for dried LEMS
analyses as this data point was clearly invalid and resulted in
a sigmoidal curve that did not fit the majority of the points,
as shown in Figure S8 in Supplementary Material. This
particular ion was excluded from the data set because a large
solvent feature obscured the parent molecular ion of p-NO2
benzylpyridinium. The derivative of the sigmoidal curve
provides the internal energy distribution, P(E), which
appears in Figure 4b for the three experimental techniques.
The mean of the internal energy distributions, GEint9 for
conventional ESI-MS, dried LEMS, and liquid LEMS
analyses were 1.62 0.06, 2.0 0.5, and 1.6 0.3 eV, respec-
tively, with full width at half maximum (FWHM) of 0.96,
1.04, and 0.50 eV, respectively. Each GEint9 uncertainty was
estimated by multiplying the G Eint 9 value by the average of
the relative standard deviations of the survival yield values
from Figure 4. The data suggest that LEMS of dried samples
deposits more energy into the system than conventional
electrospray with a 24.7% increase in GEint9. However,
analyses with liquid LEMS resulted in a comparable GEint9
to conventional ESI-MS. We note that if the mass
spectrometer had been tuned to have a low mass cutoff of
m/z 100, M+-pyridine would have been the only major
fragment included in the SY calculations, resulting in similar
internal energy distributions for all three techniques
(Figure S9 in Supplementary Material). All of the fragments
observed in the tandem MS measurements were included in
the SY calculations [43].
Investigating the High Internal Energy Deposition
in Dried Samples with LEMS
That LEMS analyses of dried BzPy ions led to more
fragmentation than in conventional ESI-MS is not unantic-
ipated as certain small molecules have been previously
observed to have additional fragmentation in the case of
laser vaporization [29, 41]. Yet, it was surprising that dried
and liquid benzylpyridinium LEMS analyses resulted in
completely different internal energy distributions. There-
fore, further experiments were conducted to elucidate a
potential mechanism of high internal energy deposition
with dried samples, including investigations concerning the
drying gas temperature, the substrate material, the sample
holder temperature, the sample drying process, and the
interaction with the laser. The results of these experiments
for p-Me and p-Cl BzPy are shown and discussed in detail
in Figure S10 in Supplementary Material. Briefly, the

1578
P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
results suggested that the drying gas temperature, the
substrate material, the sample holder temperature, and the
sample drying process did not significantly contribute to
the excess fragmentation observed in LEMS analyses of
dried benzylpyridinium salts.
Analysis of Larger Biomolecules
LEMS and ESI measurements were performed on larger
biomolecules to investigate the internal energy distribution
for systems with many more degrees of freedom. In general,
the available degrees of freedom for the molecule and
collision gas affect the resulting internal energy distribution
of the molecule [8, 49], with larger molecules having lower
average internal energies for equivalent collision energy.
The peptides leucine enkephalin (leu-enk) (556.6 Da) and
bradykinin (BK) (1060.2 Da) were analyzed from dried and
liquid conditions to determine the effect of molecular weight
on nonresonant, femtosecond vaporization. The possibility
of two-photon resonances is reduced as these peptides do not
have UV-visible absorptions around 400 nm, as seen in
Figure S13b and f in Supplementary Material. Leucine
enkephalin has been extensively used to study the internal
energy deposition using ESI-MS [8, 18, 19, 49, 50]. Dried
irinotecan HCl (623.1 Da) was also analyzed with LEMS to
examine the fragmentation as a function of molecular size
when two-photon resonant fragmentation is prevalent as this
molecule has an absorbance extending to 400 nm.
Interaction with highly intense femtosecond laser pulses
typically induces non-thermal, nonresonant, and universal
vaporization of molecules [46]. However, if a molecule has a
low order multiphoton resonance, fragmentation may occur,
particularly in the ionic state [47, 48]. UV-visible spectra were
measured for the benzylpyridinium ions to determine if the
molecules had a resonance with 800 nm light (Figure S11 in
Supplementary Material). There was no absorption at 800 nm
but there was an absorption centered at 400 nm for all of the
benzylpyridinium ions except for p-CN BzPy. Although the
UV-Vis measurement is a one-photon process, two-photon
absorption is possible since the molecules lack a center of
symmetry. UV-visible spectra were measured for the two
previously reported LEMS investigations of dried samples that
resulted in a greater amount of fragmentation than with
conventional ESI-MS [29, 41]. Tetrabutylammonium iodide,
which loses one of its butyl groups because of femtosecond
laser vaporization [41], had a broad absorption centered at
~360 nm that extends above 400 nm, as seen in Figure S12 in
Supplementary Material. Irinotecan HCl, which is studied in
detail in the next section, also had an absorption centered at
~355 nm that extends to 400 nm (Figure S13c in Supplemen-
tary Material). Two-photon excitation was possible for
tetrabutylammonium iodide and irinotecan HCl given the
broad bandwidth of the 70 femtosecond pulse and the broad
absorption features measured for the molecules. Two-photon
excitation occurred from the fringes of the 800 nm laser pulse
for tetrabutylammonium iodide and irinotecan HCl and the
lower intensities led to smaller absorption cross-sections and
reduced fragment ratios. In comparison, benzylpyridinium ions
have two-photon resonances at the most intense wavelength of
the laser pulses, leading to excessive fragmentation in dried
LEMS analyses.
If a two-photon resonance was the cause of excessive
fragmentation in the dried LEMS analyses, the question then
arises concerning why the fragmentation pattern was
different for the liquid LEMS analyses? The data suggest
that there are different mechanisms for femtosecond vapor-
ization of dried samples and liquid samples. Femtosecond
vaporization of the dried BzPy samples resulted in the
majority of the energy going into the analyte’s internal
energy, inducing a great extent of fragmentation. However,
in the analysis of liquid samples, the energy was not solely
absorbed by the analytes but also by the liquid, yielding less
fragmentation with survival yields similar to those obtained
with conventional electrospray mass spectrometry. The
liquid may also have protected the analyte from energetic
collisions with the hot drying gas, therefore decreasing the
internal energy. Conversely, the dried samples were vapor-
ized directly into the drying gas prior to capture by the
electrospray solvent.
Analysis of Leucine Enkephalin
The conventional electrospray and LEMS mass spectra
obtained for leucine enkephalin, irinotecan HCl, and
bradykinin are presented in Figure 5a–c, d–e, and f–h,
respectively, at low and high capillary exit voltages. The
corresponding breakdown curves (survival yield as a
function of capillary exit voltage) are plotted in Figure 6a–
c, respectively, for the three biomolecules. The high
resolution ESI-MS/MS mass spectra of the isolated parent
peaks and the fragmentation features included in the SY
calculations are presented in Figures S14 to S16 and
Table S2 in Supplementary Material, respectively.
The fragmentation pattern from the LEMS analysis of
dried leucine enkephalin (Figure 5b) matched the fragments
observed from conventional ESI-MS (Figure 5a), though the
LEMS analysis had a slightly higher yield of fragments. The
survival yield for dried LEMS analysis decreased as a
function of increasing ISCID energy, as seen in the
breakdown curves in Figure 6a in accord with expectations.
The extra fragmentation resulted in average decreases in
survival yield of ~15% (excluding the data point at 130 V)
and ~9% for dried LEMS analysis from glass and metal,
respectively, in comparison with conventional ESI-MS.
Leucine enkephalin does not have a two-photon resonance
but does have an absorption at 274 nm (Figure S13a in
Supplementary Material) leading to a possible three-photon
resonance occurring around 267 nm. Vaporization of leucine
enkephalin from metal or glass substrates resulted in little to
no difference in the fragmentation pattern or survival yield.
An increase in the pulse energy from 0.7 to 2.1 mJ resulted
in a ~15% decrease in the survival yield for dried leucine
enkephalin analysis from both glass and metal substrates, as

P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
1579
Figure 5. Mass spectra obtained for (a)–(c) leucine enkephalin, (d)–(e) irinotecan HCl, and (f)–(h) bradykinin using (a), (d), and
(f) conventional electrospray, (b), (e), and (g) LEMS of dried samples, and (c) and (h) LEMS of liquid samples from metal. The
plots show the fragmentation of the biomolecules at low and high capillary exit voltages of 180 and 365 V as black/gray and
red/dark red lines, respectively
seen in the inset in Figure 6a. LEMS measurements for
liquid leucine enkephalin led to less fragmentation and an
increase in the survival yield, showing a similar outcome to
benzylpyridinium analysis. Most of the major features in the
mass spectra for the LEMS liquid analysis (Figure 5c) were
solvent-related features from m/z 200 to 300. The survival
yield as a function of ISCID potential was essentially
unchanged as a function of collision energy for liquid
leucine enkephalin analysis. At lower ISCID biases, analysis
of liquid leucine enkephalin with LEMS resulted in an 8%
lower survival yield compared with ESI. However, after the
capillary exit voltage of 230 V, the survival yield decreased
for ESI-MS but not for liquid LEMS analyses, resulting in
14% less fragmentation for liquid LEMS. Increasing the
laser pulse energy did not result in a decrease of survival
yield for liquid LEMS analysis that was observed in the
dried LEMS analysis. The leucine enkephalin measurements
are consistent with the BzPy studies and further support the
idea that resonances lead to dissociation.
than with ESI-MS [29]. The mass spectra are shown in
Figure 5d–e for conventional ESI-MS and dried LEMS,
respectively. Like the leucine enkephalin study, the frag-
ments observed during LEMS analyses correspond well to
those from conventional ESI. Vaporization of irinotecan HCl
from glass resulted in lower survival yields in comparison
with those measured using a metal substrate by an average
of ~34%. Vaporization of dried p-Me BzPy (Figure S10b)
from glass also resulted in lower survival yields than from
metal, but this was not observed for p-Cl BzPy
(Figure S10e). Further experiments are needed to explain
this phenomenon. At lower capillary exit voltages (up to
230 V), LEMS analyses from a metal substrate resulted in a
9% decrease in survival yield, whereas vaporization from
glass yielded a 31% decrease in comparison with ESI. This
is consistent with the excess fragmentation in previous
LEMS experiments of irinotecan HCl. But the survival
yields for the LEMS analyses did not decrease until a
capillary exit voltage of 330 V, causing LEMS analyses to
display greater survival yields than ESI-MS at higher
collision energies. This is analogous to the LEMS measure-
ments of dried benzylpyridinium ions where the survival
yields were consistent or increased as the CID energy
increased. The increase in survival yield as a function of
Analysis of Irinotecan HCl
Mass analysis of irinotecan HCl was revisited since it
previously had been shown to fragment more with LEMS

1580
P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
throughout all the collision energies, as seen in Figure S18
in Supplementary Material. The LEMS analysis of dried
bradykinin had the second highest ratio at lower collision
energies but fell below the LEMS liquid analysis after an
ISCID potential of 230 V as vaporization of bradykinin
from aqueous solution resulted in no change in the [M+
3H+]3+/[M+2H+]2+ ratio. There was also no change in the
survival yield as a function of collision voltage for liquid
LEMS analysis, as shown in the breakdown curves
(Figure 6c), as the SY remained around 95% with a 13%
average increase compared with conventional electrospray.
The breakdown curves show that the vaporization of dried
bradykinin led to initial internal energies that were
consistent with ESI-MS until higher collision energies,
where an average decrease in survival yield of about 11%
occurred for dried LEMS. This is consistent with the
hypothesis that the mechanism of vaporization for dried
samples and liquid samples is different. The dried LEMS
SY data point at the capillary exit of 230 V was
uncharacteristically high in comparison with the rest of
the data and may be an outlier. In parallel with the leucine
enkephalin experiment, bradykinin may have a three-
photon resonance as it has an absorption centered at
256 nm (Figure S13c in Supplementary Material). The
measurements reaffirm the capability of femtosecond
vaporization to preserve the conformation of a protein
from its condensed phase as was observed previously for
aqueous lysozyme [31].
Figure 6. Breakdown curves as a function of capillary exit
voltage for the biomolecules (a) leucine enkephalin, (b)
irinotecan HCl, and (c) bradykinin using conventional ESI-
MS (black ■), LEMS of dried samples from glass (red ●) or
metal (dark red ●), and LEMS of liquid samples from metal
(green ▲). The inset in (a) shows the effect of the pulse energy
on survival yields at a capillary exit voltage of 365 V for
leucine enkephalin dried and liquid samples
Conclusions
LEMS measurements with 800 nm femtosecond laser
pulses for dried and liquid samples resulted in more
internal energy deposited into dried samples than solution-
phase samples. Analysis of liquid samples resulted in less
fragmentation, presumably through the solvent mitigating
the amount of internal energy transferred to the mole-
cules. The decrease in fragmentation may also be due to
solvent clusters protecting the molecules from the hot
drying gas in the case of LEMS of liquid analytes. A two-
photon resonance resulted in a substantial increase in
fragmentation in the analysis of dried benzylpyridinium
ions, tetrabutylammonium iodide, and irinotecan HCl. The
internal energy distributions for dried samples were
dependent on the molecular weight, with smaller mole-
cules subject to more fragmentation and lower survival
yields. Femtosecond vaporization of molecules from the
solution-phase led to internal energies that are comparable
to those obtained with conventional electrospray, regard-
less of a multiphoton resonance. To obtain the “softest”
analysis, liquid samples can be vaporized with 800 nm
femtosecond laser pulses at any pulse energy, whereas
dried samples should be desorbed with the lowest possible
pulse energies, particularly if the molecule has a multi-
photon resonance with the laser light.
capillary exit voltage is again attributed to the preferred ion
transmittance of higher mass ions as the ISCID potential
increases, causing much of the fragment intensity to be
unobserved and thereby raising the survival yield.
Analysis of Bradykinin
The mass spectra for bradykinin are presented in Figure 5f–
h, and the ion intensities are shown up to m/z 625 to
highlight the lower mass [M+2H+]2+ and [M+3H+]3+
features. The full mass spectra are shown in Figure S17 in
Supplementary Material. The trends for bradykinin are
similar to leucine enkephalin; the fragmentation features
for electrospray and LEMS of dried samples are similar,
whereas LEMS analysis of liquid samples displayed fewer
fragments and solvent-related features from m/z 200 to 300.
One major difference among the mass spectra concerns the
intensity ratio of the [M+2H+]2+ and [M+3H+]3+ features,
where ESI had the highest [M+3H+]3+/[M+2H+]2+ ratio

P. M. Flanigan IV et al.: Internal Energy Distributions of LEMS
1581
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Acknowledgments
The authors acknowledge support for this work by the
Office of Naval Research (N00014-10-0293) and the
National Science Foundation (CHE 0957694).
25. Harris, G.A., Hostetler, D.M., Hampton, C.Y., Fernandez, F.M.:
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