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PROTEINS: Structure, Function, and Genetics 28:53–58 (1997)
Gas Phase Hydrogen/Deuterium Exchange Reactions
of Peptide Ions in a Quadrupole Ion Trap Mass
Igor A. Kaltashov, Vladimir M. Doroshenko, and Robert J. Cotter*
The Johns Hopkins University School of Medicine, Department of Pharmacology, Baltimore, Maryland
Hydrogen/deuterium exchange
reactions of protonated and sodium cationized
peptide molecules have been studied in the gas
phase with a MALDI / quadrupole ion trap mass
spectrometer. Unit-mass selected precursor
ions were allowed to react with deuterated
ammonia introduced into the trap cell by a
pulsed valve. The reactant gas pressure, reaction time, and degree of the internal excitation
of reactant ions were varied to explore the
kinetics of the gas phase isotope exchange.
Protonated peptide molecules exhibited a high
degree of reactivity, some showing complete
exchange of all labile hydrogen atoms. On the
contrary, peptide molecules cationized with
sodium exhibited only very limited reactivity,
indicating a vast difference between the gas
phase structures of the two ions. Proteins 28:53–
58, 1997 r 1997 Wiley-Liss, Inc.
hydrogen-deuterium exchange reactions of biomolecular ions with quadrupole mass analyzers9 as well
as tandem sector10 and hybrid sector/quadrupole11
mass spectrometers.
Quadrupole ion trap mass spectrometers12 offer a
number of unique capabilities that make them extremely attractive to a physical chemist studying gas
phase ion-molecule reactions. To date, ion trap mass
spectrometers have been successfully applied to a
variety of analytical and physicochemical problems
related to biomolecules, ranging from peptide and
oligonucleotide sequencing13,14 to studies of thermochemistry and kinetics of ion /molecule15 and ion /
ion16 reactions. We report here on the use of a
quadrupole ion trap mass spectrometer built in our
laboratory14 to study the gas phase isotope exchange
processes of peptide ions generated by MALDI.
Key words: ion/molecule reactions; hydrogen /
deuterium exchange; ion trap;
MALDI; peptide ions
All experiments were performed on an extensively
modified Finnigan MAT (San Jose, CA) quadrupole
ion trap mass spectrometer equipped with a General
Valve (Fairfield, NJ) Series 9 pulsed valve for pulsed
gas introduction.14 Peptide ions were desorbed in an
external MALDI ion source, using the fourth harmonic (226 nm) laser pulse from a Nd:YAG laser
(Quantel International, Santa Clara, CA). These
externally produced ions were injected into the trap
cell, isolated, allowed to react with deuterated ammonia and mass analyzed using the sequence of events
shown in Figure 1.
Multiple (usually three) cycles of trapping by
injection of ions into an increasing trapping potential, followed by isotopic cluster isolation using
notched broad band excitation waveforms (periods
I-III in Figure 1), were utilized for mass selective
accumulation of peptide ions. Then, notched narrower band ‘‘stretched-in-time’’ excitation pulses (pe-
Gas phase hydrogen-deuterium exchange reactions, monitored by mass spectrometry, have been
used extensively in the past as a means for counting
labile hydrogen atoms on small organic molecules.1
With the emergence of the so-called ‘‘soft’’ ionization
techniques, such as electrospray ionization (ESI)
and matrix-assisted laser desorption/ ionization
(MALDI), gas phase isotope exchange reactions with
biological molecules have been used with intention of
providing information on the reactivity and higher
order structures of peptides and proteins in the
absence of solvent.2,3 It has been also shown that
gas-phase hydrogen-deuterium exchange reactions
of biomolecular ions have the potential for becoming
an indispensable tool for isomer distinction.4 An
attempt has been made recently to probe the threedimensional geometry of small gaseous peptide ions
by monitoring the gas-phase isotope exchange with
optically active molecules.5 Such experiments are
usually carried out on Fourier transform ion cyclotron resonance (FT ICR) mass spectrometers.2–8
Several groups have also reported studies of the
Mass Spectrometry
Contract grant sponsor: National Institutes of Health; Contract grant number R01 RR08912, Contract grant number R01
*Corresponding author: Robert J. Cotter, The Johns Hopkins
University School of Medicine, Department of Pharmacology,
725 N. Wolfe Street, Biophysics Bldg. B-7, Baltimore, MD
21205. E-mail:
Received 22 August 1996; Accepted 19 November 1996
Fig. 1.
Timing diagram of a typical hydrogen/deuterium exchange experiment.
riod IV) were applied to achieve unit mass isolation
(i.e., the isolation of the major d0 monoisotopic peak
of peptide ions).14 This step was followed by setting
the RF voltage level to achieve the value of the
Mathieu operational parameter qz 5 0.2–0.3, and
introduction of ND3 (period V) via the pulsed valve,
with variable pulse length (25–500 msec) and gas
pressure (0.025–2.5 mTorr). In the open position the
pulse valve produced a stationary level of ND3
pressure in the vacuum chamber, which was assumed to be proportional to the input gas pressure.
Because the sensitivity of an ionization Penning cold
cathode vacuum gauge (CVC, Rochester, NY) to ND3
was unknown, ND3 gas pressures were established
by comparing He gas pressure as a function of inlet
gas pressure, and assuming that the pressure after
injection will not differ strongly for different gases if
the pulse valve inlet pressure is the same. The inlet
pressure (range: 3–300 Torr) was measured with an
absolute accuracy of ca. 0.5 Torr by a piezoresistive
pressure sensor (Omega, Stamford, CT) . Constant
input pressure was maintained by using a Matheson
(Secaucus, NJ) pressure regulator for input pressure
levels higher than 50 Torr or a large volume vessel
for lower pressure levels. After gas injection, the
time for pumping out the vacuum chamber was
about 10 ms and was limited by the capacity of the
turbomolecular pump employed. Thus, a time inter-
val of 100 ms was allowed between the closing of the
pulse valve and beginning of the mass analysis stage
to permit evacuation of ND3 and eliminate its possible influence during resonance ejection (period VI).
Ions were resonantly ejected to the detector for mass
analysis at qz 5 0.34 and a mass scan rate of 1000
Da /s.
In some experiments the reacting ions were subjected to an off-resonance excitation during introduction of ND3 (period V in Fig. 1) to raise the level of
internal excitation of peptide ions. The frequency of
off-resonance sinusoidal excitation was about 5%
lower than the resonance frequency of ions and the
amplitude was within 0–15 V0-p (zero to peak). The
maximum level of excitation was determined by
observing the level at which ejection of peptide ions
or their fragments first produced an appropriate
detector signal on the oscilloscope. The duration of
the excitation was equal to that of ND3 injection
All spectra were acquired using TrapWare software, developed in-house, and processed with the
TOFWare package (ILys Software, Pittsburgh, PA).
Peptides were purchased from Sigma Chemical
Company (St. Louis, MO) and used without further
purification. Nicotinic acid (Aldrich Chemical Com-
pany, Inc., Milwaukee, WI) was used as a matrix.
Deuterated ammonia (ND3, 99.3% pure) was purchased from Isotec, Inc. (Miamisburgh, OH).
Protonated Peptide Molecules
Gramicidin S is a cyclic decapeptide (cycloLFPVOLFPVO) with two basic sites (ornithine side
chains). It has 13 potentially exchangeable hydrogen
atoms in its protonated form (5 on ornithine amine
groups and 8 amide hydrogens on the peptide backbone). Hydrogen/deuterium exchange with protonated gramicidin S (MH1), without prior selection of
the monoisotopic peak d0, shows a significant shift
(,5 Da) in the isotopic distribution (Fig. 2). While
deconvolution of the isotopic distribution can be used
to deduce the exact number of exchanged hydrogens,8 this procedure would not lead to reliable
results in our case since changes in the isotopic
distribution of desorbed ions were observed shortly
after beginning the experiment even in the absence
of pulsed ND3 introduction. This is apparently caused
by absorption of residual ND3 molecules on the
matrix surface followed by isotope exchange on the
probe (or in the ablation plume region during the
desorption process). This difficulty was overcome by
selecting the d0 monoisotopic peak (see experimental
section) before introducing ND3 into the trap cell,
and greatly simplified the interpretation of the data.
Figure 3 shows the results of the hydrogen/deuterium exchange reactions of the d0 peak at different
reaction times and ND3 pressure. The maximum
achievable number of exchanged hydrogen atoms in
these experiments is six, suggesting that the rather
fast isotope exchange events at the protonation site
are followed by intramolecular H/D exchange (or
‘‘scrambling’’) at slower rates.
McLafferty and coworkers3 have observed a significant increase in the number of exchanged hydrogen
atoms as a result of elevating the internal energies of
multiply charged protonated ions by heating the
trapped ions within the FTICR cell with either IR
laser irradiation or collisional activation. Contrary
to that, Cheng and Fenselau10 observed a decrease in
the isotope exchange rates of peptide ions resulting
from the collisional activation of ions in a third
field-free region of a four-sector mass spectrometer.
Similar effects have been observed for smaller molecules.17,18 We have explored the influence of the ion
internal energy on the exchange rates and extent
using off-resonance excitation of trapped ions.
The accurate calculation of the steady-state internal energy of ions undergoing off-resonance excitation in the ion trap is an elaborate procedure.19 To
characterize ion excitation quantitatively, we use
another parameter, ion collision energy, which can be
controlled easily by adjustment of excitation amplitude and frequency, and estimated by using a pseudopotential well approximation.14 Since the steady-
Fig. 2. Hydrogen/deuterium exchange of protonated gramicidin S without prior selection of d0 monoisotopic peak: natural
isotope abundance (a) and reaction products resulting from a 300
msec pulse of ND3 at 0.45 mTorr (b).
state amplitude of the ion oscillation during the
off-resonance excitation is directly proportional to
the amplitude of the excitation signal, the upper
limit of collision energy of ions undergoing excitation
in the ion trap, may be estimated as energy of
circular motion of ions, by using the pseudopotential
well approximation14:
Ecoll 5
mM(ptnr0)2 (V02p)2
max 2
where m and M are the masses of the ion and the
target gas molecule, respectively; r0 5 1 cm is the
radius of the ring electrode, n is the frequency, and
V0-p is the amplitude of the dipole excitation field.
Vo-pmax is the excitation amplitude at which total ion
signal disappearance occurs. The influence of the
off-resonance excitation on the rates of hydrogen/
deuterium exchange reactions has been studied in
the present work by varying the amplitude of the
excitation signal between the zero value and the
dissociation/ejection threshold at a fixed frequency.
Collisional activation led to a significant increase in
the number of exchanged hydrogen atoms, as shown
on Figure 4. The horizontal axis of this graph shows
the amplitude of excitation. The total disappearance
of ionic signal at this frequency was observed at
Fig. 4. Influence of the internal energies on the isotope
exchange: relative abundances of isotopic peaks dn plotted as
functions of the excitation voltage; X, d0; c, d5; N, d7; M, d8.
Excitation frequency is 113.6 kHz and ND3 pressure is 0.045
Fig. 3. Hydrogen/deuterium exchange of protonated gramicidin S with prior selection of d0 monoisotopic peak. a: Unreacted d0
peak. b: Reaction products resulting from a 50-msec pulse of ND3
at 0.027 mTorr. c: 500 msec pulse of ND3 at 0.027 mTorr. d: 250
msec pulse of ND3 at 2.5 mTorr.
V0-pmax 5 17 V. We have observed a complete substitution of ‘‘active’’ hydrogen atoms (i.e., the d13 peak has
been detected) at the exchange reaction conditions
(reaction time and ND3 pressure) identical to those
shown on Figure 3d. To achieve such high level of
exchange, an off-resonance excitation at 113.6 kHz
with the amplitude of 8 V (the upper limit of collision
energy ca. 0.5 eV according to equation 1) has been
applied during the ND3 introduction into the cell. It
must be noted, however, that the internal energy
increase caused by the collisional activation does not
lead to a monotonic increase in the extent of deuterium incorporation into the peptide ion, as illustrated on Figure 4. The visible decrease in the
isotope exchange rate at high excitation level is very
likely to be caused by the significant decrease of the
formation rate and lifetime of [MH1...ND3] complexes.10,17,18
These observations are consistent with the following model of the hydrogen/deuterium exchange in
the gas phase: complexation of the protonation site
with the reactant gas–isotope exchange–dissociation
of the complex–intramolecular isotope exchange, the
latter being a rate-determining step. The first three
steps are likely to proceed via the classical mechanism usually described with a double well potential
diagram.1,10,11 The last step depends on the internal
energy of the peptide ion as well as its flexibility. The
fact that all active hydrogen atoms may be exchanged in a singly protonated gramicidin S ion
suggests that this peptide is quite flexible in the gas
phase, despite significant steric constrains imposed
by the cyclic backbone. Another possible mechanistic
model explaining the effects observed includes intramolecular charge transfer as a fourth (rate-limiting)
step in the scheme described above. This proton
transfer to a ‘‘new’’ location within the peptide is
followed by complexation-exchange-dissociation sequence.
Results similar to that observed for protonated
gramicidin S molecules were also obtained for other
protonated peptide ions such as bradykinin and
Sodium Cationized Molecules
Earlier results of ion mobility measurements of
Na1 adducts of polyethylene glycol molecules suggested that these cationized polymers form rather
compact and rigid structures in the gas phase, which
are ‘‘cemented’’ by the alkali metal ion.20 If this holds
true for peptides as well, one might expect to see
significantly slower isotope exchange reactions in
the gas phase for peptide molecules cationized with
sodium. In order to check this hypothesis we performed a series of experiments with gramicidin S
cationized with sodium, similar to those described
earlier for protonated gramicidin S.
Although sodiated gramicidin S has 12 possibly
exchangeable hydrogen atoms, incorporation of only
two deuterium atoms has been detected (Fig. 5). Use
of off-resonance excitation not only failed to increase
the number of exchanged hydrogen atoms, but actually led to a decrease in the relative abundances of d1
and d2 species. These surprising results might be
explained by the assumption (see above) that complexation by the sodium cation results in a more rigid
peptide backbone that in this case significantly
limits (or completely eliminates) intramolecular hydrogen/deuterium exchange. The fact that only two
hydrogen atoms readily undergo the exchange suggests that these two atoms are probably located in
close proximity to the charge site. It is reasonable to
assume that the charge site is the amino group of the
side chain of one of the ornithine residues. The
complexation is very likely to be a result of interaction of sodium cation with the carbonyl oxygen atoms
of the peptide backbone. Although this ‘‘charge solvation’’ may also occur in the case of protonated gramicidin S,7 the size difference between H1 and Na1 ions
would result in a much ‘‘less rigid’’ structure. Interestingly, smaller peptides (such as leucine-enkephalinamide) exhibited even slower hydrogen /deuterium
exchange when cationized with Na1. Alternatively,
an explanation based on the intramolecular charge
transfer, can also be used to justify the much slower
exchange rate of the cationized peptide, since the
mobility of Na1 ion must be significantly lower than
that of a proton. This model, however, leaves open
the question on why only two exchanges are observed (it appears that gramicidin S has several
candidate sites for Na1 attachment).
We have demonstrated that a quadrupole ion trap
mass spectrometer with an external MALDI source
is a valuable tool for providing information on both
Fig. 5. Hydrogen/deuterium exchange of sodium cationized
gramicidin S molecules MNa1 with prior selection of d0 monoisotopic peak: a: unreacted d0 peak; b: reaction products resulting
from a 250 msec pulse of ND3 at 2.5 mTorr.
detailed kinetics and energetics of hydrogen/deuterium exchange reactions in the gas phase. The
results of experiments reported in the present work
are consistent with a four-step mechanism of deuterium incorporation into peptide ions. This mechanism includes (i) complexation of the protonation
site with the reactant gas, (ii) isotope exchange
within the complex, (iii) dissociation of the complex
and (iv) intramolecular isotope exchange. Increase
in the internal energy of the peptide ion decreases
the rate of the first step. At the same time, it leads to
an increase of the rate of the last step, most likely
due to conformational changes. It appears that significantly elevated internal energy is a prerequisite
for a complete exchange of all ‘‘active’’ hydrogens, at
least in the case of singly charged ions. A tremendous
difference in the rates and extent of hydrogen /
deuterium exchange of protonated and metal ion
cationized peptides suggests that the three-dimensional structures of these ions in the gas phase have
significant differences. Cationization of a peptide
with alkali metal ions leads to a formation of a rather
rigid three-dimensional structure in the gas phase,
precluding the intramolecular isotope exchange. An
alternative interpretation, based on the mobile charge
model, may also be used to account for the observed
This work was supported by grants from the
National Institutes of Health (R01 RR08912 and R01
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