International Journal of Analytical Mass Spectrometry and Chromatography, 2013, 1, 90-94
Published Online December 2013 (http://www.scirp.org/journal/ijamsc)
http://dx.doi.org/10.4236/ijamsc.2013.12011
Open Access IJAMSC
Formation of Mercury(II)-Glutathione Conjugates
Examined Using High Mass Accuracy Mass Spectrometry
Zachary Fine1, Troy D. Wood2*
1Department of Chemical and Biological Engineering, University at Buffalo, State University of New York, Buffalo, USA
2Department of Chemistry, University at Buffalo, State University of New York, Buffalo, USA
Received October 12, 2013; revised November 9, 2013; accepted December 10, 2013
Copyright © 2013 Zachary Fine, Troy D. Wood. This is an open access article distributed under the Creative Commons Attribution
License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
In accordance of the Creative Commons Attribution License all Copyrights © 2013 are reserved for SCIRP and the owner of the
intellectual property Zachary Fine, Troy D. Wood. All Copyright © 2013 are guarded by law and by SCIRP as a guardian.
ABSTRACT
Maternal exposure to Hg(II) during pregnancy has been identified as a potential causal factor in the development of
severe neurobehavioral disorders. Children with autism have been identified with lower reduced glutathione
(GSH)/oxidized glutathione (GSSG) ratios, and GSH is known to strongly bind Hg(II). In order to gain insight into the
mechanism by which GSH binds Hg(II), high resolution mass spectrometry coupled with tandem mass spectrometry
was utilized to examine the conjugation process. While the 1:1 Hg(II):GSH conjugate is not formed immediately upon
mixing aqueous solutions of Hg(II) and GSH, two species containing Hg(II) are observed: the 1:2 Hg(II):GSH conju-
gate, [(GS)2Hg + H+], and a second Hg(II)-containing species around m/z 544. Interestingly, this species at m/z 544 de-
creases in time while the presence of the 1:1 Hg(II):GSH conjugate increases, suggesting that m/z 544 is an intermediate
in the formation of the 1:1 conjugate. Experiments using the high mass accuracy capability of Fourier transform ion
cyclotron resonance (FT-ICR) mass spectrometry coupled to an electrospray ionization source indicate that the interme-
diate species is [GSH + HgCl]+, and not the 1:1 conjugate [Hg(GSH) – H + 2H2O]+ postulated in previous literature.
Further confirmation of [GSH + HgCl]+ is supported by collision of induced dissociation experiments, which show neu-
tral loss of HCl from the intermediate and loss of the N- and C-terminal amino acids, indicating binding of Hg(II) at the
Cys residue.
Keywords: Glutathione; Mercury; FT-ICR; Mass Spectrometry; Tandem Mass Spectrometry
1. Introduction
Glutathione (GSH, γGlu-Cys-Gly) is a tripeptide with a
reactive thiol group found in relatively high intracellular
concentrations, and it is the primary regulator of cellular
redox homeostasis paired with its disulfide (GSSG) [1,2].
Some heavy metals such as Hg(II) are toxic to cells be-
cause of their ability to deplete GSH [3]. The binding
constant of Hg(II) to GSH is very large as measured by
polarographic [4] and nuclear magnetic resonance [5]
methods. Zalups has expertly reviewed the role of thiol-
containing proteins, including GSH, and their interac-
tions with Hg(II) in uptake, accumulation, transport, and
toxicity [6].
Electrospray ionization mass spectrometry (ESI-MS)
has been used previously to examine the conjugation of
Hg(II) with GSH [7-11]. The studies of Rubino et al.
provided insight into the stoichiometry of binding be-
tween Hg(II) and GSH, with particular attention on the
dissociation behavior of the conjugates under collision-
ally induced dissociation (CID) conditions by tandem
mass spectrometry (MS/MS) [9,11]. Interestingly, it was
found that the 1:1 Hg(II):GSH conjugates require sig-
nificantly higher collisional activation energy than the
1:2 Hg(II):GSH or the 1:1 Hg(II):GSSG conjugates,
suggesting strong coordination of Hg(II) at the carboxy
terminus of GSH [9]. Burford et al. examined Hg(II)
conjugation to GSH in both positive ion and negative ion
ESI-MS, and also found the existence of 1:1 and 1:2
Hg(II):GSH conjugates [10]. Burford et al.’s results were
replicated by positive ion ESI-MS and it was found that
Hg(II):GSH conjugates spiked into plant extracts could
be recovered and detected [8]. Of particular note was the
publication of detailed isotopic insets, which proved that
*Corresponding author.

Z. FINE, T. D. WOOD 91
Hg(II) was indeed conjugated to GSH [8]. A mercury-
glutathione conjugate with 1:3 Hg(II):GSH stoichiometry
has been shown in negative ion mode ESI-MS [7].
Here, we report on the conjugation between Hg(II) and
GSH using high resolution mass spectrometry. Our mo-
tivation for this investigation stems from three items.
First, lower GSH:GSSG ratios in the plasma of children
with autism have been found, which have been related to
oxidative stress [12]. Second, as detailed thoroughly in
reviews [13,14], there is an extensive evidence that ex-
posure to mercury leads to neurological conditions. Third,
all the previous ESI-MS reports were done on relatively
low-resolution mass spectrometers, and we felt that an
investigation using high resolution mass spectrometry
would be useful to validate earlier studies and resolve
existing ambiguities in interpretation of the ESI mass
spectra from Hg(II):GSH conjugates. ESI coupled to
Fourier transform ion cyclotron resonance mass spec-
trometry (ESI-FTICR) is used in the current investigation.
The high resolution mass spectrometry data provide a
keen insight into the mechanism by which Hg(II):GSH
conjugates form.
2. Experimental
All high resolution mass measurements were acquired
using a Bruker Daltonics (Billerica, MA) 12 tesla So-
lariX FT-ICR mass spectrometer equipped with an ESI
source. Mercuric nitrate (J. T. Baker, Phillipsburg, NJ)
and reduced glutathione (Sigma-Aldrich, St. Louis, MO)
were prepared as separate aqueous solutions, each at 10
μg/mL. These were then mixed in a 1:1 volume:volume
ratio and then loaded into a syringe for infusion into the
ESI source. ESI was performed at 5.4 kV using N2 nebu-
lizer gas (2.9 L/min) by infusing the sample mixture via
syringe pump at 3.0 µL/min and applying N2 drying gas
(2.8 L/min, 200˚C) to desolvate the droplets. Ions were
accumulated in the external quadrupole for 100 ms be-
fore transfer through the external ion optics to the
FT-ICR trap. Ions were trapped in an Infinity cell (front
trap plate 0.6 V, back trap plate 0.8 V) [15] operating at
~1 × 10−9 mbar, and excited and detected in broadband
mode as positive ions over m/z 98.3-3000 using 2 MB
time domain data sets; the data was zero-filled once and
displayed in the magnitude mode. The resulting FT-ICR
mass spectra are from the summation of 100 individual
scans. Collision induced dissociation (CID) experiments
were performed by isolation of the isotopic cluster in the
mass-selective quadrupole and dissociation in the colli-
sion cell with argon gas (~1 × 10−3 mbar). All isotope
distributions were simulated using the proposed ionic
formulas using the IsotopePattern utility in Bruker Dal-
tonics’ Compass software package.
3. Results and Discussion
Upon mixing 10 μg/mL aqueous solutions of mercuric
nitrate and GSH, ESI-FTICR mass spectra were collected
immediately and the resulting data is shown in Figure
1(a). One of the primary analytical advantages of FT-
ICR is its ability to measure ionic masses with unparal-
leled accuracy [16]. Accurate mass measurements con-
firm the presence of (M + H)+ for both reduced glu-
tathione (308.09317 Da, +2.1 mDa error) and oxidized
glutathione (613.1633 Da, +4.1 mDa error). In addition,
two peaks due to dioctyl phthalate, a common plasticizer,
are observed, which include (M + H)+ at 391.2870 Da
(+2.7 mDa error) and (M + Na)+ at 413.2690 Da (+2.8
mDa error) which serve as internal mass calibrants. As
shown in the inset of Figure 1(a), a very weak peak (less
than 1% relative abundance) due to an isotopic cluster
with its most abundant peak at m/z 815.1349 is attributed
to [(GS)2Hg + H+] (+5.2 mDa); this species has been
observed in previous reports of Hg(II) conjugated to
GSH [8-11]. A 1:1 Hg(II):GSH conjugate is not observed
immediately after mixing, but a curious isotopic cluster
centered around m/z 544 having an isotopic pattern con-
sistent with a single Hg(II) is observed. Only once has
this species been reported in positive ion ESI mass spec-
tra, and that was collected at low resolution; hence, the
identity of the species remained ambiguous, although it
was speculated that the species responsible “might be a
Hg-GS cluster with two water molecules associated with
it” [8]. However, as discussed below, high mass accuracy
and MS/MS reveal another composition involving Hg(II).
Interestingly, with time the 1:1 Hg(II):GSH conjugate
appears, and after four days of incubation at room tem-
perature, it becomes by far the dominant Hg(II)-contain-
ing species in the ESI-FTICR mass spectrum (Figure
1(b)); [(GS)2Hg + H+] is still present, but remains weak.
Intrigued by the fact that the species containing Hg(II)
around m/z 544 decreases with time while the 1:1
Hg(II):GSH conjugate around m/z 508 increases with
time, we compared the high mass accuracy for the m/z
544 conjugate and its isotopic distribution against that
previously predicted [8]. Figure 2(a) represents an inset
of the isotopic distribution measured around m/z 544 by
ESI-FTICR, and bears a striking similarity to the isotopic
distribution reported for this ion by Krupp et al. in Figure
2d of their paper [8], suggesting these are in fact due to
the same ionic species. However, this experimen-
tally-derived result does not match well with the theo-
retically-predicted isotopic distribution for the postulated
[Hg(GSH) – H + 2H2O]+ (Figure 2(b)) [8]. Accurate
mass measurement of the nominal m/z 544 peak by ESI-
FTICR is 544.0263 Da, which further indicates [Hg(GSH)
– H + 2H2O]+ cannot be the identity of this ionic species.
However, this accurate mass measurement is in close
Open Access IJAMSC

Z. FINE, T. D. WOOD
Open Access IJAMSC
92
Figure 1. ESI-FTICR mass spectra of aqueous solutions of Hg(II) mixed with GSH (a) immediately after mixing (t = 0) and (b)
after 4 days incubation at room temperature. The inset in Figure 1(a) represents the magnified region of m/z 810 - 820, indi-
cating the presence [(GS)2Hg + H+], which is confirmed by accurate mass analysis.
Figure 2. (a) Inset of ESI-FTICR mass spec trum of aque ous solution s of Hg(II) mixed with GSH after four days incubation at
room temperature around m/z 544; (b) Simulated isotope distribution for [Hg(GSH) – H + 2H2O]+; (c) Simulated isotope
distribution of [GSH + HgCl]+.

Z. FINE, T. D. WOOD 93
agreement with another possible formula corresponding
to [GSH + HgCl]+; a simulation of its theoretical isotope
distribution is shown in Figure 2(c). The experimental
ESI-FTICR data in Figure 2(a) agrees well not only with
the theoretical isotopic distribution, but also with the
predicted ionic mass (544.0217 Da) with only 4.6 mDa
error.
To validate that chlorine was indeed present in this
species, we conducted MS/MS using CID. The entire
isotopic cluster centered about m/z 544 was mass-se-
lected in the source quadrupole of the SolariX FT-ICR
and dissociated with argon gas in the collision cell before
transport to the FT-ICR ion trap. CID experiments were
conducted over the range 8 - 16 V, but the product ions
generated only showed small differences in relative
abundance and not in the actual product ions produced,
so only the 12 V CID result is shown in Figure 3. CID
confirms that chlorine is present in the formula for m/z
544; neutral loss of 35.9764 Da is observed, correspond-
ing to loss of HCl. The neutral loss of 111.0079 Da is
due to the loss of glycine hydrochloride from the C-ter-
minus of GSH, which is followed by subsequent neutral
losses of H2O and NH3, respectively. The peak at m/z
379.0032 Da is consistent with the loss of the N-terminal
μ-glutamic acid residue [17,18] from the m/z 508 product
ion, while m/z 306.0756 corresponds to GS+; these results
completely support a previously reported dissociation
mechanism of the 1:1 Hg(II):GSH conjugate [9].
It is curious to consider how an aqueous solution of
mercuric nitrate would produce this intriguing intermedi-
ate species. It should be noted that commercial mercuric
nitrate does contain low levels of chloride impurities.
Thus, GSH may form the intermediate by the following
reaction:

2
GSH HgClGSH HgClCl

 
(1)
In order to have such a composition with Hg(II) bound
at the thiol, to maintain the overall mass of GSH, the N-
terminus must be in its protonated form. If so, then the
neutral loss of HCl can be explained as shown in Scheme
1. The product ion would then match the zwitterionic
structure proposed for m/z 508 [9]. Additional support for
this interpretation is derived from the fragmentation ob-
served by CID, where the loss of the N-terminal and
C-terminal amino acids from the conjugate occurs with-
out the loss of the mercury atom, implying that it is in-
deed covalently bound to the central Cys residue.
4. Conclusion
Previous low-resolution ESI-MS of Hg(II) conjugates
with GSH was unable to establish conclusively the iden-
tity of an isotopic cluster centered about m/z 544, al-
though it was postulated that such an ion might be a 1:1
conjugate with two associated water molecules. Here, the
Figure 3. CID(12V) of the m/z 544 cluster generated by ESI-
FTICR.
Scheme 1. Fragmentation of [GSH + HgCl] + intermediate.
high mass accuracy capability of FT-ICR coupled to an
ESI source of Hg(II)-GSH mixtures establishes that the
isotopic cluster is [GSH + HgCl]+; further confirmation is
established through the observed isotopic ratios, consis-
tent with a species containing single Hg and Cl atoms,
and CID, which shows neutral loss of HCl upon colli-
sional activation of the precursor ion. Based on kinetics
studies, the conjugate apparently forms immediately
upon mixing. However, the conjugate species [GSH +
HgCl]+ observed is unstable, and eventually decays into
[GSH – H + Hg]+, an isotopic cluster centered about m/z
508, which has been reported previously. Thus, the high
mass accuracy capability of FT-ICR provides insight into
the mechanism of formation of a prominent organomer-
cury thiol.
5. Acknowledgements
The authors would like to thank Heather Rudolph, Will
Friesen, Charmion Cruickshank, and Kevin Quinn for
helpful discussions. Acquisition of the FT-ICR mass
spectrometer was supported by the National Institutes of
Health (S10-RR029517).
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Z. FINE, T. D. WOOD 94
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