JOURNAL OF MASS SPECTROMETRY

J. Mass Spectrom. 34, 930–941 (1999)

Analysis of Phytochelatin–Cadmium Complexes

from Plant Tissue Culture Using
Nano-electrospray Ionization Tandem Mass
Spectrometry and Capillary Liquid
Chromatography/Electrospray Ionization
Tandem Mass Spectrometry

Ten-Yang Yen,1,2 Joseph A. Villa2 and Jane G. DeWitt2 *

1 Biomedical Research Mass Spectrometry Facility, Department of Chemistry and Biochemistry, San Francisco State
University, San Francisco, California 94132-4163, USA
2 Department of Chemistry and Biochemistry, San Francisco State University, San Francisco, California 94132-4163, USA

Phytochelatins (PCs, also known as class III metallothioneins), a family of sulfhydryl-rich peptides with the
formula (g-GluCys)n Gly(Pcn , n = 2–11), are induced in plants, yeast and fungi exposed to heavy metals,
and are thought to detoxify metals by forming PC–metal complexes. Although PCs have been detected,
PC–metal complexes have not been well characterized. In this work, nano-electrospray ionization tandem mass
spectrometry (nano-ESI-MS/MS) and capillary liquid chromatography/electrospray ionization tandem mass
spectrometry (capillary LC/ESI-MS/MS) methods were used to analyze PC–Cd complexes isolated from Datura
innoxia, also known as Jimsonweed, cell culture exposed to Cd. With nano-ESI-MS/MS and capillary LC/ESI-
MS/MS we could simultaneously detect the presence of PCs and PC–Cd complexes from plant cell extracts,
unambiguously identify these species and elucidate the nature of individual PC–Cd complexes. Phytochelatins
with n = 3–6 were detected, as were PC–Cd complexes with PC3 , PC4 and PC5 . This is the first study to report
the size and nature of native PC–Cd complexes from plant tissue samples. These results demonstrate that the
direct analysis of plant extracts using nano-ESI-MS/MS and capillary LC/ESI-MS/MS methods is simple and
sensitive to the range of PCs and PC–Cd complexes in plants. Hence these methods open up new opportunities
for further quantitative analysis of PCs and PC–metal complexes in cell culture and plant systems to understand
the relationship between the biosynthesis of these compounds and metal tolerance. Copyright  1999 John
Wiley & Sons, Ltd.
KEYWORDS: phytochelatins; cadmium; nanospray; electrospray ionization; liquid chromatography/tandem mass
spectrometry; tandem mass spectrometry

INTRODUCTION agricultural plants, there is a growing concern about the

Cadmium, a widely used metal in the metal plating indus-
-GluCys/n Gly.PCn , n D 2–11/, are induced
try, nickel-cadmium batteries and a contaminant in phos-
phate fertilizers, is known to be toxic to plants and humans
(a probable human carcinogen).1,2 Acute exposure to Cd
vapor in humans can result in pulmonary irritation and
long-lasting impairment of lung function. Chronic expo-
sure to Cd affects the function of the kidney and lung.
Since one of major intake of Cd by humans is from
* Correspondence to: J. G. Dewitt, Department of Chemistry and
Biochemistry, San Francisco State University, San Francisco, Cali-
fornia 94132-4163, USA.
E-mail: dewitt@sfsu.edu
Contract/grant sponsor: National Center for Research Resources.
Contract/grant sponsor: Office of Research on Minority Health,
National Institutes of Health; Contract/grant number: 5 P20 RR11805.
levels of Cd in soil.
Phytochelatins (PCs, also known as class III metalloth-
ioneins), a family of sulfhydryl-rich peptides with the
formula .
in plants exposed to heavy metals, and function in
the detoxification of metals.3 – 7 Plants exposed to Cd2C
synthesize high levels of PCs and form PC–Cd com-
plexes which remove free Cd2C from solution, protecting
metal-sensitive enzymes. Studies on Cd-tolerant and Cd-
sensitive lines of the plant Datura innoxia, also known as
Jimsonweed, show that both lines accumulate similar lev-
els of PC–Cd complexes and synthesize similar amounts
of PCn .n D 2–4/ at similar rates, but the onset of synthe-
sis of PC–Cd complexes is more rapid in metal-tolerant
plant lines than metal-sensitive lines.16 Additionally, Cd is
associated with higher PCn .n D 4 and 5) complexes in the
tolerant lines than the sensitive lines based on gel filtration
chromatography and high-performance liquid chromato-
graphic (HPLC) results. These studies suggest that it is

CCC 1076–5174/99/090930– 12 $17.50 Received 28 January 1999

Copyright  1999 John Wiley & Sons, Ltd. Accepted 1 June 1999
 ANALYSIS OF PHYTOCHELATIN-Cd COMPLEXES BY NANO-ESI-MS/MS AND LC/ESI-MS
the assembly of the PC–Cd complexes that is important
for metal tolerance in D. innoxia rather than the ability to
synthesize phytochelatins.
Several analytical methods such as chromatographic
separation (gel filtration or HPLC) coupled with UV
detection, flame atomic absorption spectrometry (AAS),
radioactive labeling and inductively coupled plasma mass
spectrometry (ICP-MS) have been used to analyze PCs
and PC–Cd complexes.5 – 7 These methods, however, do
not directly provide structural information about PC–Cd
complexes. The PC–Cd complexes are generally isolated
by gel filtration chromatography as a broad fraction of
Mr D 3000–6000. Analysis of the PC content in this frac-
tion involves acidification .pH  2/, which destroys the
metal complex,5 followed by HPLC to resolve the individ-
ual PCn components in the PC–Cd fraction. The presence
of a range of PCn s as determined by HPLC suggests that
a range of PCn –Cd complexes may be formed; however,
gel filtration does not have sufficient resolution to provide
information about the types or nature of PC–Cd com-
plexes present in the PC–Cd fraction. If the formation of
PC–Cd complexes is the key factor in metal tolerance,
then a technique with high mass resolution is required to
be able to detect complexes that may differ in size by only
a few hundred mass units. Additionally, a high-sensitivity
technique is required to be able to detect compounds
that may be present at low levels in biological samples.
The innovation of electrospray ionization mass spectrom-
etry (ESI-MS),8 a powerful technique for the analysis
of proteins and peptides with a sensitivity of low fem-
tomole .10 15 mol/ to high attomole .10 18 mol/,9 has
greatly enhanced our capability to analyze organometallic
complexes,10 including the interaction between a peptide
or protein and a metal ion.11 – 17 So far, few studies of
the use of ESI-MS in the analysis of PCs have been
reported,16 and the direct analysis of PC–Cd complexes
has not been explored.
In this study, we applied nano-electrospray tandem
mass spectrometry (nano-ESI-MS/MS)18,19 and capillary
liquid chromatography/electrospray ionization tandem
mass spectrometry (capillary LC/ESI-MS/MS)20 for the
analysis of PCs and PC–Cd complexes induced in
Datura innoxia tissue culture exposed to Cd. Using these
techniques, PCn s with n D 3–6 and Cd complexes with
each of these PCs were detected.
-GluCys/5 Gly (10 3 M) or
EXPERIMENTAL
-GluCys/5 Gly (5 ð 10 4 M)–Cd (2.5 ð 10 4 M) mix-
Materials
HPLC-grade water, methanol (MeOH) and acetonitrile
(Mallinckrodt, Paris, KY, USA) were obtained from
Fisher Scientific (Fair Lawn, NJ, USA). Cadmium chlo-
ride .CdCl2 / was purchased from Aldrich (Milwaukee,
WI, USA). The chemical standard of .
-GluCys/5 Gly was gain control to avoid the space charge effects.22 The full-
synthesized by Commonwealth Biotechnologies (Rich-
mond, VA, USA), and was used without further purifica-
tion. Other chemicals utilized, such as acetic acid (AcOH)
and trifluoroacetic acid (TFA), were of analytical grade,
and were obtained from Fisher Scientific.
Copyright  1999 John Wiley & Sons, Ltd.
931
Isolation of the PC–Cd complex from D. innoxia
A metal-tolerant D. innoxia tissue culture cell-line was
obtained from Dr Paul Jackson of Los Alamos National
Laboratory (Los Alamos, NM, USA). Cultures of 50 ml
of D. innoxia tissue were grown according to published
methods6 and exposed to 250 µM CdCl2 for 24 h. A
detailed procedure for the isolation of PCs and PC–Cd
complexes is described elsewhere.21 In brief, cell culture
and media were transferred into 50 ml sterilized conical
tubes and centrifuged at 800 g for 2 min. After centrifuga-
tion, the packed cells (8.5 ml) were collected. Cells were
re-suspended, washed in 20 ml of ice-cold buffer (10 mM
Tris–HCl, pH 7.4, 10 mM KCl and 1.5 mM MgCl2 ) and
centrifuged again. The buffer was decanted and the cells
were washed twice more with the same buffer. Cells
were homogenized on ice in extraction buffer (10 mM
Tris–HCl, pH 7.4, 10 mM KCl, 1.5 mM MgCl2 , 20 mM
ˇ-mercaptoethanol) using a hand-held homogenizer. The
homogenate was centrifuged at 10 000 g for 15 min and
the supernatant was removed and stored at 4 ° C.
PCs and PC–Cd complexes were isolated from the
supernatant by gel filtration chromatography using a Bio-
Rad (Hercules, CA, USA) Econo LC system. A 20 ml
volume of supernatant was loaded on to a Bio Gel P-6
column (100 ð 2.5 cm i.d.) and eluted using Tris–HCl
(50 mM, pH 7.8) at a flow-rate of 0.6 ml min 1 . The elu-
ate was monitored at 254 nm and 7.5 ml fractions were
collected. The Cd content for each fraction was deter-
mined using a Unicam 929 atomic absorption spectrome-
ter (ThermoJarrel Ash, Franklin, MA, USA) at 228.8 nm.
Fractions that both absorbed at 254 nm and contained Cd
by AAS were pooled (67.5 ml) and concentrated using
an Amicon stirred-cell concentrator (Millipore, Bedford,
MA, USA) with a 500 MW cut-off membrane. A vol-
ume of 200–500 µl of concentrated extract was dried
using a SpeedVac (Savant Instruments, Hicksville, NY,
USA) and re-suspended in either 20 µl of water or 50%
methanol–water with 3% acetic acid prior to the ESI-
MS analysis (estimated concentration 1.6 ð 10 3 –4.0 ð
10 3 M Cd2C as PC–Cd complex).
ESI-MS/MS analyses
ESI-MS/MS analysis was performed using a Finnigan
(San Jose, CA, USA) LCQ ion trap mass spectrome-
ter. The chemical standard .
.
tures were prepared and infused into the ESI source at
3 µl min 1 . A positive voltage of 4–5 kV was applied to
the needle and the temperature of the stainless-steel heat-
ing capillary was maintained at 180–250 ° C. An N2 sheath
flow (65 scale) was applied to stabilize ESI signal. The
voltage at the exit of the heating capillary and the tube
lens was held at 13 and 5 V, respectively, to minimize the
source-induced dissociation and optimize the ESI signal of
the analyte. The ion injection was controlled by automatic
scan mass spectrum was acquired from m/z 150 to 2000.
MS/MS and MSn experiments were executed with a rela-
tive collision energy of 32–39% (1.60–1.95 Vpeak to peak
of r.f. resonance excitation voltage). The charge state and
isotopic abundance of the analyte were determined by an
J. Mass Spectrom. 34, 930–941 (1999)
932 T.-Y. YEN, J. A. VILLA AND J. G. DEWITT
LCQ high-resolution scan (zoom scan) with an isolation
window of 10 Da.
The nano-ESI source was constructed according to
the method published by Davis and Lee.23 Briefly, the
nanospray needle, an uncoated fused-silica tip (360 µm
o.d., 75 µm i.d. with 8 or 30 µm tip) (New Objective,
Cambridge, MA, USA), was connected to one end of a
-GluCys/n Gly with n D 2–5. This work
micro-Tee connector (Upchurch, Oak Harbor, WA, USA),
and a platinum wire (350 µm o.d.) was connected to the
vertical end of the Tee to apply voltage. The other end of
the Tee was connected to a 25 µl syringe using a fused-
silica capillary tube (20 cm ð 50 µm i.d.). Sample was
loaded into the syringe and the flow-rate was controlled
using the syringe pump on the LCQ. The needle assem-
bly was mounted on a xyz multi-axis translational stage
(Newport, Irvine, CA, USA) to position the needle, and a
charge-coupled device (CCD) camera with a monitor was
used to guide the needle direction. Normally, the needle
tip was positioned 1 mm away from the center of the pin-
hole of the heating capillary. A voltage of 0.8–1.3 kV
was sufficient to maintain a stable ESI signal. In this
work, we used a nanospray needle with a 30 µm tip size
-GluCys/5 Gly.PC5 / standard, the
(0.2–0.3 µl min 1 ) because this size of needle is easy to
use and is not to prone to clogging. Therefore, the needle
can be re-used several times. Using a smaller (8 µm) tip
size of the nanospray needle can further reduce the sample
flow-rate to less than 50 nl min 1 , but it is easily clogged.
Capillary LC/MS/MS analysis was conducted using
a Hewlett-Packard (Palo Alto, CA, USA) Model 1050
HPLC system coupled to the LCQ. The HPLC system was
operated at a flow-rate of 0.3 ml min 1 . The mobile phase
was split before the injector by a Tee-connector. One
end of the Tee was connected to a capillary C18 column
(150 ð 0.18 mm i.d.) Nucleosil, 5 µm particle size) and a
flow-rate of 1.5 µl min 1 was established. The capillary
column was packed according to the methods described
by Davis et al.,24 with the modification that a stainless-
steel frit (2 µm) was used instead of a PVDF membrane
frit, and HPLC fittings were used for the connection to
eliminate the use of epoxy. Volumes of 1–5 µl of the
D. innoxia extract were injected via a Valco (Houston,
TX, USA) injector mounted on the LCQ, and eluted from
the column using 0.01 or 0.1% TFA in water (mobile
phase A) and 0.1% TFA in acetonitrile (mobile phase B).
PC–Cd complexes were eluted using a two-step linear
gradient of 1–3% B in the first 10 min, followed by
3–30% B in the next 30 min. The eluate from the capillary
column was connected via a fused-silica capillary (100 µm
i.d., 165 µm o.d.) to the ESI source. The capillary LC/ESI-
MS/MS analysis was accomplished using an automated
data acquisition procedure, in which a cyclic series of
three different scan modes were performed. The data
acquisition was conducted using the full-scan mode to
obtain the most intense peak (signal >1.5 ð 105 counts)
as the precursor ion, followed by a high-resolution scan
to determine the charge state of the precursor ion and an
MS/MS scan to determine the structural fragment ions of
the precursor ion.
RESULTS AND DISCUSSION
Previous investigations of the formation of PC–Cd
complexes in D. innoxia 6 showed that a shift to higher
Copyright  1999 John Wiley & Sons, Ltd.
molecular mass of the Cd-containing fraction from
cell lysates occurred with time of exposure to Cd2C ,
suggesting the formation of a Cd complex of some
sort. Acidification of the Cd-containing fraction followed
by HPLC analysis showed the presence of four peaks
and amino acid analysis of those peaks confirmed their
identity as .
demonstrated that the biosynthesis of phytochelatins was
induced in D. innoxia upon exposure to Cd2C , and that
PCs were involved in the Cd complex, but the size,
stoichiometry and nature of the PC–Cd complexes was
not elucidated. Nano-ESI/MS/MS should provide the
resolution needed to detect and identify PCs and PC–Cd
complexes rapidly with greater sensitivity and with less
sample preparation.
Analysis of a (g-GluCys)5 Gly standard
To test the applicability of the nano-ESI-MS/MS method
for the analysis of PCs and PC–Cd complexes, a com-
mercially synthesized .
largest PC found in D. innoxia,6 was analyzed. The nano-
ESI spectrum of PC5 (1 mM) in 3% AcOH–50% MeOH
is dominated by the singly charged [M C H]C ion at
m/z 1236.2, which matches the elemental composition
of PC5 .C42 H65 N11 O22 S5 / with a monoisotopic molecular
mass of 1235.3, as shown in Fig. 1(a). The high-resolution
(zoom) scan mode of PC5 demonstrated that not only
the analyte charge state, but also the distribution of ana-
lyte isotopic peaks [Fig. 1(b)] could be resolved. The
detected ion abundance ratio of the monoisotopic ion (m/z
1236.1), [M C H]C , over the ion abundance ratio of the
[M C H C 1]C or [M C H C 2]C peaks closely matched
the theoretical value.25 From five measurements includ-
ing that shown in Fig. 1(b), we obtained average ratios of
[M C H C 1]C /[M C H]C and [M C H C 2]C /[M C H]C
of 0.49 š 0.02 and 0.34 š 0.02, respectively, in good
agreement with the theoretical values25 of 0.57 and 0.42,
respectively. The chemical identity of an analyte can be
determined using the high-resolution MS technique.26,27
With the resolution R.m/mFWHM / D 6000 obtained in
our ion trap mass spectrometer, the ion formation of
PC–Cd complexes can be unambiguously determined by
their isotopic peak distribution (discussed below). It is also
notable in the spectrum that ions of m/z 1234.3 and 1235.3
were detected. The difference of 2 Da between the ion at
m/z 1236.3 [M C H]C and m/z 1237.3 [M C H C 1]C and
m/z 1234.3 and m/z 1235.3, respectively, suggests that a
disulfide bond may form between two Cys residues in
the PC5 monomer (formation would be accompanied by a
loss of 2H). The disulfide bond was confirmed by MS/MS
analysis (data not shown). The tandem mass spectrum of
PC5 at m/z 1236 [Fig. 1(c)] revealed that both b- and y-
terminus28 ions were the dominant fragment ions, which
served to confirm the identity of the analyte.
Analysis of Cd complexes of (g-GluCys)5 Gly
To study PC5 –Cd complexes, we prepared a solution of
PC5 (0.5 mM) and Cd2C (0.25 mM) in 3% AcOH–50%
MeOH and analyzed the solution using the nano-ESI-
MS/MS technique. This solution system optimized the
detection of the analyte signal while maintaining the
J. Mass Spectrom. 34, 930–941 (1999)
 ANALYSIS OF PHYTOCHELATIN-Cd COMPLEXES BY NANO-ESI-MS/MS AND LC/ESI-MS 933

Figure 1. Nano-ESI-MS/MS of . -GluCys/5 Gly in 50% MeOH 47% H2 O 3% AcOH. The full-scan mass spectrum (a), the high-resolution
scan at m/z 1236 (b) and the tandem mass spectrum of the precursor ion at m/z 1236 with an isolation width m D 10 and a relative
collision energy of 35% (c) are presented.

Copyright  1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 930–941 (1999)
934 T.-Y. YEN, J. A. VILLA AND J. G. DEWITT
Figure 1. (continued ).
PC–Cd complex. The intensities of PC5 and its Cd com-
plexes were greatly reduced in solution with the Cd2C
concentration ½0.3 mM. Solutions with a ratio of PC5
to Cd of 0.5 to 0.25 mM gave the optimal ESI signal
for PC5 and PC5 –Cd complexes. Spectra are shown in
Fig. 2(a), (b) and (c). Cd2C is a thiophilic metal ion; the
formation of a phytochelatin–Cd2C complex is therefore
expected to involve the cysteine thiol groups of phy-
tochelatin with the loss of 2HC for each coordinated Cd2C
ion. PC5 could accommodate up to two Cd2C ions involv-
ing four of the five cysteine residues. The mass spectrum
of the PC5 –Cd solution [Fig. 2(a)] shows three PC5 –Cd
complexes at m/z 1346, 1458 and 1568, corresponding to
PC5 –Cd, PC5 –Cd2 and PC5 –Cd3 , respectively. The m/z
values for the first two complexes are consistent with the
loss of 2HC for each Cd2C , supporting the formation of
a covalent PC–Cd complex, while the m/z value for the
third complex is consistent with a third Cd2C associated
electrostatically with the PC5 –Cd2 complex. As shown
in the inset zoom scan of PC5 in Fig. 2(a), it is also
notable that there is an increase in the ion peak intensities
at m/z 1234.2 and 1235.2 compared with those peaks in
Fig. 1(b). This suggests that cysteine residues in PC5 are
more readily oxidized to intramolecular disulfide bonds
owing to the presence of excess Cd2C in solution.
Figure 2(b) is a representative spectrum obtained from
an average of 50 zoom scans at m/z 1342–1352 to
show the isotope pattern of the PC5 –Cd complex. The
isotopic peak distribution of the PC5 –Cd complex is
complicated owing to the isotopic distribution for PC5
[see Fig. 1(b)] coupled to the isotopic distribution of Cd
(106 Cd, 1.25%; 108 Cd, 0.9%; 110 Cd, 12.5%; 111 Cd, 12.8%;
Copyright  1999 John Wiley & Sons, Ltd.
112
Cd, 24.1%; 113 Cd, 12.2%; 114 Cd, 28.7%; 116 Cd, 7.5%).
To ascertain that the detected isotopic distribution pat-
tern is reproducible, a replicate of six measurements was
performed, and the mean and standard deviation of the
observed intensity for each isotopic peak were calcu-
lated (each measurement represents an average of 50–80
zoom scans from experiment). These results are summa-
rized in Table 1. Assignment of the isotope peaks (see
Table 1) indicates the presence of two types of PC5 –Cd
complexes, one with [PC5 C Cd-2 C H]C for the cysteine-
coordinated Cd2C complex, and the other with the for-
mula [PC5 C Cd-4 C H]C for a PC5 –Cd complex with
an intramolecular disulfide bond involving two additional
cysteine residues. Table 1 compares the expected ion
signal intensities calculated for the [PC5 C Cd-2 C H]C
complex and the [PC5 C Cd-4 C H]C complex with the
observed intensity for each m/z peak obtained from exper-
imental measurements. The m/z values for many of the
peaks in Fig. 2(b) could be assigned to the presence of
either [PC5 C Cd-2 C H]C or [PC5 C Cd-4 C H]C , but the
peak at m/z 1342.2 and 1343.2 can only be assigned
to the presence of [PC5 C Cd2C -4 C H]C with 110 Cd and
111
Cd isotopes, confirming the presence of complexes
of this type. Similarly, the peak at m/z 1350 is mostly
attributed to the [PC5 C Cd-2 C H]C with 116 Cd, suggest-
ing that both types of complexes are present. Additionally,
the calculated intensities for just [PC5 C Cd-2 C H]C or
[PC5 C Cd-4 C H]C do not match the observed intensi-
ties. If peak intensities are calculated for a mixture of the
complexes consisting of 45% [PC5 C Cd-2 C H]C C 55%
[PC5 C Cd-4 C H]C , good agreement between the calcu-
lated intensities and the observed intensities for each m/z
J. Mass Spectrom. 34, 930–941 (1999)
 ANALYSIS OF PHYTOCHELATIN-Cd COMPLEXES BY NANO-ESI-MS/MS AND LC/ESI-MS 935

Figure 2. Nano-ESI-MS/MS of a mixture of . -GluCys/5 Gly (0.5 mM) and Cd2C (0.25 mM) in 50% MeOH 47% H2 O 3% AcOH. The
full-scan mass spectrum (a) is presented with an inset zoom scan of m/z 1236 showing the presence of oxidized PC5 at m/z 1234 and
1235. The zoom scan of m/z 1347 (PC5 Cd complex) (b) corresponds to a mixture of Cd complexes with reduced and oxidized PC5 . The
tandem mass spectrum of PC5 Cd complex (c) with settings of the precursor ion at m/z 1346, isolation width m D 10 and a relative
collision energy of 35% shows that Cd2C remains with most of fragments generated (signified by an asterisk). Most of the unassigned
ions are either bŁ 18 or yŁ 18.

Copyright  1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 930–941 (1999)
936 T.-Y. YEN, J. A. VILLA AND J. G. DEWITT

Figure 2. (continued ).

Table 1. Comparison of the observed ion peaks and intensities with the expected peaks and
intensities for [PC5 Y Cd−2 Y 1]Y and [PC5 Y Cd−4 Y 1]Y and for a mixture of
the two complexes
Expected ion signal intensities (%)
Observed ion 45% [PC5 C Cd-2 C H]C C
m/z signal intensitya (%) [PC5 C Cd-2 C H]C [PC5 C Cd-4 C H]C 55% [PC5 C Cd-4 C H]C

1342.2 18 š 4 3 26 18
1343.2 34 š 8 1 41 26
1344.2 70 š 7 26 75 60
1345.2 69 š 3 41 68 63
1346.2 100 75 100 100
1347.2 70 š 7 68 54 68
1348.2 81 š 11 100 48 81
1349.2 44 š 6 54 21 41
1350.2 36 š 7 48 11 31
1351.2 9š3 21 3 12
a
Values of the observed ion signal intensities represent the mean and standard deviation
obtained from six different measurements.

peak seen in Fig. 2(b) is obtained. We would like to point
out that this calculation is not intended to serve as a
quantitative assay but rather to indicate that the isotope
distribution of the PC5 –Cd complex has contributions
from both [PC5 C Cd-2 C H]C and [PC5 C Cd-4 C H]C
ions. The observation of the [PC5 C Cd-4 C H]C ion also
confirms the presence of the intramolecular disulfide bond
in samples with the PC5 model compound.
We performed MS/MS analysis on the precursor ion
at m/z 1346 to explore the structure of the PC5 –Cd
complex, and the spectrum is shown in Fig. 2(c). The
isolation window was set at š5 Da with respect to the
precursor ion, m/z 1346, to accommodate the isotopic
peaks of the PC5 –Cd complexes. These settings generated
reproducible tandem mass spectra. The dominant fragment
ions are the [M C H 18]C ion .bŁ 11 / and the [M C H-
Glu]C ion .yŁ 10 /, where M is the combination of .PC5 C
Cd-2) and .PC5 C Cd-4) as described above, and the
asterisk indicates the presence of Cd in the fragment.
The signal intensities of the fragment ions of the PC5 –Cd
complexes are lower than those of the fragment ions from
the MS/MS analysis of PC5 shown in Fig. 1(c). Similar

Copyright  1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 930–941 (1999)
 ANALYSIS OF PHYTOCHELATIN-Cd COMPLEXES BY NANO-ESI-MS/MS AND LC/ESI-MS
observation of a decrease in the fragmentation efficiency
-GluCys/n Gly–
of metal complexes has been reported by others.13,14 The
bn - and yn -terminal ions (n D 6–10 and b5 Ł as well as
b4 Ł ) clearly contain the Cd isotopic pattern. Although the
tandem mass spectrum has an extensive fragmentation
pattern, interpretation of the data to resolve the exact
location of Cd coordination in the PC5 –Cd complexes
remains a complicated task, since fragmentation from
-GluCys/4 Gly (m/z
either the N-or C-terminus of the peptide results in Cd-
-GluCys/5 Gly (m/z 1236), the signal
bound peptide fragments. More likely, these yŁ and bŁ
-GluCys/4 Gly–Cd complex ion is about
fragments suggest a mixture of Cd binding sites in PC5 .
-GluCys/5 Gly–Cd complex ion,
Analysis of PC–Cd complexes from plant tissue
culture
The goal of this research is to use nano-ESI-MS/MS
in the direct analysis of PC–Cd complexes isolated
from D. innoxia tissue culture. To that end, the PC–Cd
fraction was isolated using gel filtration chromatogra-
-GluCys/4 Gly–Cd complex with the pre-
phy and concentrated. A 500 µl aliquot of the con-
centrated PC–Cd fraction was dried and dissolved in
-GluCys/4 Gly–Cd complex are either the
20 µl of 50% MeOH–3% AcOH prior to analysis by
nano-ESI-MS/MS. The full scan is shown in Fig. 3(a),
-GluCys/4 Gly–Cd
and the peaks seen at m/z 540.1, 772.2, 1004.2, 1236.2
-GluCys/5 Gly–Cd complex from D. innoxia is sim-
and 1468.2 are assigned as .
-GluCys/n Gly with n D ilar to that of the standard .
-GluCys/5 Gly–Cd complex
2, 3, 4, 5 and 6, respectively. The inset zoom scan of PC5
(m/z 1231–1241) in Fig. 3(a) shows that the ion signal
intensities at m/z 1234 and 1235 are much lower than
those at m/z 1236 and 1237 in the cell extract sample com-
pared with the PC5 –Cd model sample [see Fig. 2(a)]. This
indicates that oxidized forms of PC5 from D. innoxia were
not formed, owing to the presence of ˇ-mercaptoethanol
in the extraction buffer which protects sulfhydryl groups
from oxidation. Similar results were seen for PC3 , PC4
and PC6 (data not shown).
The presence of PC6 in D. innoxia has not been
reported previously.6 The structures of the PC3 –PC6 pep-
tides were confirmed by MS/MS analysis and the tandem
mass spectrum for PC6 (m/z 1468) is shown in Fig. 3(b).
From the assigned y-terminal ions, the data clearly show
that PC6 fragments by losing successive
-GluCys units, analysis, we employed capillary LC/ESI-MS to analyze
yielding the fragment ions .
-GluCys/n GlyC with n D the PCs and the PC–Cd complexes from D. innoxia. A
2–5, at m/z 540, 772, 1004 and 1236. This was the
same fragmentation pattern as seen for the PC5 standard
[Fig. 1(c)].
The relative distributions of the different PCs deter-
mined by nano-ESI-MS/MS is different from the dis-
tribution determined by HPLC reported previously.6 In
Fig. 3(a), the most abundant form of the PCs is PC4
followed by PC5 , PC3 and PC6 , assuming similar ion-
ization efficiencies for each PC. The level of PC2 may be
reduced owing to loss of that peptide during concentration
of the PC–Cd fraction with the 500 Mr cut-off membrane.
By HPLC, PC2 was present in the greatest amount after
24 h of growth, with decreasing amount of PC3 , PC4 and
PC5 , suggesting that the longer chains are synthesized
more slowly and accumulate with time of exposure.6 The
greater abundance of PC4 compared with PC3 seen by
nano-ESI-MS/MS suggests that there may not be a corre-
lation between time of Cd exposure and accumulation of
-GluCys/n Gly peaks was affected
PCs of large size; however, a time course study is needed
to elucidate this issue further.
The nano-ESI spectrum for the D. innoxia sample
[Fig. 3(a)] also contains rich information about the
Copyright  1999 John Wiley & Sons, Ltd.
937
PC–Cd complexes induced by Cd. The .
Cd complexes for n D 4 and 5 are observed at m/z 1116
and 1348, with both peaks containing the complex isotopic
distribution pattern noted for the PC5 –Cd standard
(data not shown). The signal intensities of the PC–Cd
complexes are much weaker .<10%/ than those of the
PCs without Cd. In contrast to the much higher signal
intensity of the [M C H]C ion for .
1004) than that for .
intensity of the .
equal to that of the .
suggesting similar rates of complex formation. Moreover,
unlike the PC5 standard, PC–Cd2 and PC–Cd3 complexes
were not detected in D. innoxia. The lack of complexes
of other than 1 : 1 PC : Cd stoichiometry probably reflects
the lower concentration of Cd and the competition for Cd
by other PCn s in vivo.
Figure 3(c) shows the tandem mass spectrum gener-
ated from the .
cursor ion m/z 1115. As expected, the dominant frag-
ments of the .
N- or C-terminal ions containing Cd (yŁ or bŁ ). The
MS/MS fragmentation pattern of the .
or .
shown in Fig. 2(c), except for the detailed isotope dis-
tribution pattern of yŁ or bŁ . The differences seen are
due to the lack of oxidized forms of the PCn s and their
Cd complexes from D. innoxia due to the presence of
ˇ-mercaptoethanol (described for Fig. 3(a) above). The
isotope pattern of yŁ and bŁ fragments from the PC5 –Cd
model [Fig. 2(c)] is composed of a mixture of .PC5 C Cd-
2CH/ and .PC5 C Cd-4CH/, while the isotope pattern
of yŁ and bŁ fragments from the D. innoxia extracts
[Fig. 3(c)] is mainly composed of .PC4 C Cd-2CH/.
LC/ESI-MS analysis of PC–Cd fraction from
D. innoxia
To confirm the result obtained from nano-ESI-MS/MS
custom-packed capillary C18 column (0.18 mm i.d.) was
used to enhance the sensitivity of LC/MS. The smaller
diameter of the LC column gives a higher analyte peak
concentration for an equal amount of injected analyte.20
The ESI response depends on sample concentration;
samples with higher concentration generate more intense
signals.29 We found that the sensitivity of LC/MS using
the 0.18 mm i.d. column was twofold higher than that
using the 0.3 mm i.d. column. For the capillary LC/ESI-
MS/MS analysis of D. innoxia extracts, 1–5 µl of the
sample solution were injected and analyzed. A linear
gradient of water with 0.1 or 0.01% TFA and acetonitrile
with 0.1% TFA was employed to elute the PCs and the
PC–Cd complexes. The elution profile of phytochelatins
is dependent on their molecular mass, with the lowest
Mr PCs eluting first and the highest Mr PCs eluting last.
Each PC–Cd complex co-eluted with the corresponding
PC. The shape of the .
by the percentage of TFA in the mobile phase. In a mobile
phase without TFA, the PCs did not separate according
to molecular mass, and the peak shape was broadened.
Using a gradient of 0.1% TFA in water (a) and 0.1% TFA
J. Mass Spectrom. 34, 930–941 (1999)
938 T.-Y. YEN, J. A. VILLA AND J. G. DEWITT

Figure 3. Nano-ESI-MS/MS of the Datura innoxia tissue culture sample. The full-scan mass spectrum from the D. innoxia extracts with
an inset zoom scan of m/z 1236 .PC5 / (a) show low levels of oxidized PC5 as evidenced by low peak intensity at m/z 1234 and 1235. The
tandem mass spectrum at m/z 1468 (b) corresponds to . -GluCys/6 Gly with settings of isolation width m D 10 and a relative collision
energy of 39%. The tandem mass spectrum for . -GluCys/4 Gly Cd complexes at m/z 1115 with isolation width m D 10 and a relative
collision energy of 35% (c) are presented. The asterisk denotes ions that contain Cd. Most of the unassigned ions are either bŁ 18
or yŁ 18.

Copyright  1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 930–941 (1999)
 ANALYSIS OF PHYTOCHELATIN-Cd COMPLEXES BY NANO-ESI-MS/MS AND LC/ESI-MS
Figure 3. (continued ).
in acetonitrile (b) resulted in a decrease in the signal for
the PC–Cd complexes compared with a TFA content of
0.01%, suggesting the loss Cd from the complex under
-GluCys/n Gly–Cd complex to
conditions of low pH .2/, as has been noted before.5,30
-GluCys/n Gly are similar to those results by obtained
We have optimized the LC/ESI-MS/MS separation of the
PC–Cd complexes using a two-step linear gradient from
1 to 3% B in the first 10 min and from 3 to 30% B
-GluCys/3 Gly–Cd complex (shown in Fig. 4(e), m/z
in the next 30 min (A D 0.01% TFA–water, B D 0.1%
TFA–acetonitrile). The elution peak shape of PCs using
0.01% TFA in water was slightly broadened compared
-GluCys/3 Gly–Cd2 complex. These results indi-
with 0.1% TFA in water, but the signal for the PC–Cd
complexes was greater, thus minimizing the loss of the
PC–Cd complexes.
Figure 4(a)–(d) show the reconstructed ion chro-
matograms (RIC) of the .
-GluCys/n Gly peptides .n D
3–6/ from D. innoxia at m/z 772, 1004, 1232 and 1468.
The capillary LC/ESI-MS/MS data were acquired using
an automatic collection sequence of full scan, high-
resolution scan and MS/MS scan. The jagged peak shape
observed in Fig. 4(a)–(d) is due to the intensity differ-
ence between the full-scan and the high-resolution scan,
or between the high-resolution scan and the MS/MS scan
(see Experimental). The lower molecular mass peptides
-GluCys/5 Gly–Cd complexes generated using nano-
PC3 [Fig. 4(a)] and PC4 [Fig. 4(b)] are eluted earlier
on the C18 capillary column, and some of these pep-
tides elute in the solvent front at 4 min. The peaks at
18 and 21 min in Fig. 4(a) are the fragment ion peaks
generated in the MS/MS analysis of .
-GluCys/4 Gly was dramatically reduced. This will allow us to perform
[m/z 1004, Fig. 4(b)] and .
-GluCys/5 Gly [m/z 1236, the series of full-scan, high-resolution scan and MSn scan
Fig. 4(c)], respectively. The most intense peak in the
capillary LC/ESI-MS/MS analysis for D. innoxia sam-
ple is PC4 [Fig. 4(b)], similar to the result from the
nano-ESI-MS/MS analysis. Both PC4 –Cd and PC5 –Cd
Copyright  1999 John Wiley & Sons, Ltd.
939
were detected using capillary LC/ESI-MS/MS, and had
the same retention time as the corresponding PCn .
The signal ratio of the .
.
nano-ESI-MS/MS (data not shown). Additionally, the cap-
illary LC/ESI-MS/MS analysis detected the presence of
a .
884), which was not detected using nano-ESI-MS/MS, as
well as a peak at m/z 994, consistent with the presence
of a .
cate that capillary LC/ESI-MS/MS is capable of detecting
the presence of low-level analytes in a complex mixture
whose signal is suppressed using nano-ESI-MS/MS.
CONCLUSION
Employment of nano-ESI-MS/MS has greatly enhanced
our ability to analyze PCs and PC–Cd complexes.
For example, the [M C H]C ion response of the
.
ESI-MS/MS at 0.05–0.2 µl min 1 was three times more
intense than that generated by conventional ESI at
3 µl min 1 . Because of the lower flow-rate used for nano-
ESI-MS/MS, the amount of sample needed for the analysis
required to obtain structural information from a limited
amount of biological sample.
This work demonstrates that the direct analysis
of PCs and PC–Cd complexes from plant extracts
J. Mass Spectrom. 34, 930–941 (1999)
940 T.-Y. YEN, J. A. VILLA AND J. G. DEWITT

Figure 4. Analysis of the Datura innoxia tissue culture sample using capillary LC/ESI-MS/MS. The reconstructed ion chromatograms
(RIC) of . -GluCys/3 Gly at m/z 772 (a), . -GluCys/4 Gly at m/z 1004 (b), . -GluCys/5 Gly at m/z 1236 (c) and, . -GluCys/6 Gly at m/z 1468
(d) and the full-scan mass spectrum for the peaks between 14 and 16 min (e) are presented.

Copyright  1999 John Wiley & Sons, Ltd. J. Mass Spectrom. 34, 930–941 (1999)
 ANALYSIS OF PHYTOCHELATIN-Cd COMPLEXES BY NANO-ESI-MS/MS AND LC/ESI-MS
by nano-ESI-MS/MS and capillary LC/ESI-MS/MS
is simple and capable of detecting the PCs and
PC–Cd complexes produced in plants in response
to metal stress. The nano-ESI-MS/MS trace revealed
comprehensive information about the range of PCs and
PC–Cd complexes induced in D. innoxia tissue culture
exposed to Cd. The capillary LC/ESI-MS/MS method
is complementary to the nano-ESI-MS/MS method and
is useful for detecting low-intensity peaks such as the
.
GluCys/3 Gly–Cd complex, which was only detected
by the capillary LC/ESI-MS/MS method. The nano-ESI-
MS/MS and the capillary LC/ESI-MS/MS methods can
be used to determine directly the size, stoichiometry
and number of PCn –Cd complexes in plant tissue
extracts, information that is not available by conventional
methods of investigation.4 The resolution of gel filtration
chromatography, used to follow the production of the
PC–Cd complex, is insufficient to separate the individual
PCn –Cd complexes present in D. innoxia cells, but this
resolution is easily achieved using nano-ESI-MS/MS
and capillary LC/ESI-MS/MS. The detection of PC–Cd
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Acknowledgements
This research was supported by a Research Infrastructure in Minority
Institutions (RIMI) award from the National Center for Research
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