A R T I C L E S
Tian et al.
instrument, a 200 µM solution of cysteine dissolved in a 3:1 (v/v)
mixture of methanol and water with enough lithium hydroxide to make
the solution slightly basic was pumped at a flow rate of 10 µL/min
into a Z-spray (Micromass) ESI source. The extracted ions were
accumulated in a hexapole to build up signal and then were injected
into a radio frequency (rf) only quadrupole ion guide and transported
into the FTMS cell, where they were cooled by a pulse of argon and
trapped by the dynamically assisted gated trapping technique.7 The M
- 1 ion of cysteine was isolated using an arbitrary waveform excitation,
and its reactivity was effectively the same regardless of which
instrument was used.
Hydrogen-deuterium exchange reactions were monitored as a
function of time with four different deuterated reagents which were
leaked into the system at pressures between 1 × 10-7 and 5 × 10-7
torr for a minimum of 5 h immediately prior to carrying out
experiments. The effective deuterium content of the deuterated reagents
was determined by examining the H/D exchange of isophthalate (m-
C6H4(CO2H)CO2-), since it reacts at the collision-controlled rate with
all of the deuterated alcohols used in this work and incorporates only
one deuterium atom. As a result, the final d1/d0 ratio provides the useful
deuterium content of the exchange reagent in the reaction region.8 This
was found to be 90-100 %D in each case. Pseudo-first-order
bimolecular H/D exchange reaction rate constants were determined from
the decay of the reactant (d0) ion and the pressure of the alcohol in the
reaction region; no correction was made for the small amount of protic
material. In all cases, the linear fit of the experimental data gave r2 g
0.998. Apparent and site-specific rate constants also were computed
using a kinetic program developed by He and Marshall,9 and in this
case the deuterium content of the reagent and reverse processes (e.g.,
d1 f d0) were accounted for in the rate determination. As apparent
and site-specific rate constants give essentially the same information
when the former is statistically corrected, only the latter rates are
presented herein, but all of the rate constants, and graphical examples
of the data fits, are given in the Supporting Information.
An equilibrium acidity measurement was carried out on cysteine in
the dual-cell instrument by measuring forward and reverse proton-
transfer rate constants with chloroacetic acid. In one direction, the
amount of cysteine added to the cell needs to be determined. This is
difficult because it is not very volatile and was added via the solid
probe inlet. As a result, the sample is added ∼1 cm from the reaction
region and ∼1 m from the ionization gauge used to measure its pressure.
This leads to a pressure differential which must be addressed. It was
dealt with by measuring the reaction rates of cysteine with hydroxide
and fluoride ions one after the other since these reactions safely can
be assumed to occur at the collision limit.10 The resulting pressure
correction factors were 7.45 and 7.32, and the average of these two
values was employed. The B3LYP/aug-cc-pvdz dipole moment for
cysteine of 4.58 D was used in computing average dipole orientation
(ADO) rates,11 but the resulting acidity is not very sensitive to this
value (i.e., dipole moments spanning from 4.0-5.0 D alter ∆H0acid by
only up to 0.1 kcal mol-1).
basic (1% NH4OH) 49.5:49.5 H2O/MeOH were directly infused at flow
rates of 10-20 µL/min into an LCQ DECA ion trap mass spectrometer.
Solution and ion-focusing conditions were adjusted in order to maximize
the formation of proton-bound dimer ions of the form [A--H+-Bi-]-,
where A- is deprotonated cysteine and Bi- is the deprotonated reference
acid. The proton-bound dimer ions were isolated at qz ) 0.250 V with
a mass-width adjusted to maximize ion signal while still maintaining
isolation. The isolated ions were allowed to undergo collision-induced
dissociation with the background helium atoms. The ratio of the
deprotonated reference acid to deprotonated cysteine was obtained by
performing an activation amplitude scan from 0% to 100% in steps of
2. The final ion ratios are averages of at least three scans obtained on
several different days.
Gas-phase acidities and entropy contributions were obtained from
the extended kinetic method that has been described in detail
elsewhere.15-18 The final version of the extended kinetic method takes
the form
∆H0 - ∆H0
∆S0
∆H0A - ∆H0
∆S0
I
Bi-
Bi
av
Bi
av
A
ln
≈
-
+
-
( )
IA-
RTeff
RTeff
R
R
A plot of ln[I(Bi-)/I(A-)] versus ∆H0 - ∆H0av is generated, where
B
i
∆H0 is the gas-phase acidity of reference acid i and ∆ H0 is the
B
av
i
average acidity of the set of reference acids. This procedure is repeated
for data obtained at each of the collision energies. Rather than using
the traditional form of the extended kinetic method in which a second
plot is generated, the orthogonal distance regression (ODR) method of
Ervin and Armentrout was used to extract the gas-phase acidity and
entropy contribution.19 This approach has been shown to give more
realistic uncertainties for the derived thermochemical values. The ODR
procedure uses ion ratios from n reference acids and m different collision
energies and is used to create m best-fit lines to the data, forcing them
to cross at a single isothermal point. The x-coordinate of the isothermal
point is ∆H0 - ∆H0av, and the y-coordinate is ∆S0A/R, where ∆H0
A
A
and ∆S0 are the gas-phase acidity and deprotonation entropy for
A
cysteine, respectively. Final uncertainties are obtained from Monte Carlo
simulations in which random noise is generated within user-defined
ranges of the uncertainties in the gas-phase acidity of each reference
acid and in the experimental ion ratios. For these studies, the uncertainty
in the reference acids was taken to be (2 kcal mol-1 and the uncertainty
in the ln(ratio) values was (0.05.
Computational Methods. Geometry optimizations were carried out
on cysteine and its conjugate base using the Becke three-parameter
hybrid exchange and Lee-Yang-Parr correlation density functional
(i.e., B3LYP)20 and Dunning’s augmented correlation-consistent dou-
ble-ú basis set (i.e., aug-cc-pvdz)21 on workstations at the Minnesota
Supercomputer Institute running Gaussian 03.22 Both thiolate and
carboxylate ions were examined, and a variety of conformations were
probed. In an attempt to ensure that the most stable structures were
located, Monte Carlo calculations using the MMFF force field were
run using Spartan 04 on a Macintosh PowerPC G4 computer.23 The 10
most stable species for cysteine and its M - 1 ion were reoptimized
using density functional theory. Vibrational frequencies subsequently
were computed, and unscaled values were used to provide zero-point
energies (ZPEs) and thermal corrections to 298 K. G3B3 calculations,24
An electrospray ionization-quadrupole ion trap instrument was used
to carry out kinetic acidity measurements via procedures that have been
given in detail elsewhere.12-14 Briefly, dilute (∼5-10 × 10-4 M)
solutions of cysteine and one of a series of reference acids in slightly
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R. D. Rapid Commun. Mass Spectrom. 2001, 15, 1558-1561.
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(10) Bartmess, J. E.; McIver, R. T., Jr. In Gas Phase Ion Chemistry; Bowers,
M. T., Ed.; Academic Press: New York, 1979; Vol. 2, pp 87-121.
(11) Su, T.; Bowers, M. T. Int. J. Mass Spectrom. Ion Phys. 1973, 12, 347-
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Yang, W. T.; Parr, R. G. Phys. ReV. B 1988, 37, 785-789.
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(22) Pople, J. A.; et al. Gaussian 03; Gaussian, Inc.: Pittsburgh, PA, 2003.
(23) Spartan ’04 for Macintosh; Wavefunction, Inc.: Irvine, CA, 2005.
(24) Baboul, A. G.; Curtiss, L. A.; Redfern, P. C.; Raghavachari, K. J. Chem.
Phys. 1999, 110, 7650-7657.
(12) Kuntz, A. F.; Boynton, A. W.; David, G. A.; Colyer, K. E.; Poutsma, J. C.
J. Am. Soc. Mass Spectrom. 2002, 13, 72-81.
(13) Schroeder, O. E.; Andriole, E. J.; Carver, K. L.; Poutsma, J. C. J. Phys.
Chem. A 2004, 108, 326-332.
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5404 J. AM. CHEM. SOC. VOL. 129, NO. 17, 2007