Determination of sulfur and selected trace elements in metallothionein-like proteins using capillary electrophoresis hyphenated to inductively coupled plasma mass spectrometry with an octopole reaction cell
The determination of sulfur in biologically relevant samples such as metalloproteins is described. The analytical methodology used is based on robust on-line coupling between capillary electrophoresis (CE) and octopole reaction cell inductively-coupled plasma mass spectrometry (ORC–ICP–MS). Polyatomic ions that form in the plasma and interfere with the determination of S at mass 32 are minimised by addition of xenon to the collision cell. The method has been applied to the separation and simultaneous element-specific detection of sulfur, cadmium, copper, and zinc in commercially available metallothionein preparations (MT) and metallothionein-like proteins (MLP) extracted from liver samples of bream (Abramis brama L.) caught in the river Elbe, Germany. Instrumental detection limits have been calculated according to the German standard procedure DIN 32645 for the determination of sulfur and some simultaneously measured trace elements in aqueous solution. For sulfur detection limits down to 1.3 μg L−1 (34S) and 3.2 μg L−1 (32S) were derived. For the other trace elements determined simultaneously detection limits ranging from 300 ng L−1 (58Ni) to 500 ng L−1 (66Zn, 55Mn) were achieved. For quantification of sulfur and cadmium in a commercially available MT preparation under hyphenated conditions the use of external calibration is suggested. Finally, the need for proper sample-preparation technique will be discussed.
KeywordsInductively coupled plasma mass spectrometry Capillary electrophoresis Hyphenation Collision cell Metallothionein Speciation Biomolecules
Since their discovery in 1957, metal-binding proteins such as metallothioneins (MT) have been at the focus of research in biology and medicine because of their various functions in connection with the transport, storage, and detoxification of both essential and toxic trace elements in different organisms. One characteristic feature of all MT is the occurrence of Cys-xAA-Cys tri-peptide sequences, where xAA represents an amino acid residue other than cysteine. As a result, mammalian Class 1 MT contains 20 cysteine residues which cause the high affinity of this protein group for up to 12 mono (Cu+, Ag+) and seven divalent (Hg2+, Cd2+, Zn2+) metal ions. Due to genetic polymorphism, MT may be found as different isoforms which differ slightly in their amino acid sequences and therefore have different chromatographic and electrophoretic properties [1, 2, 3, 4].
The most challenging analytical task, besides extraction and separation of the different MT isoforms, is the development and application of element- and isoform-specific detection and quantification techniques due to the different biological functions suggested for different isoforms . Solving these problems requires efficient chromatographic or electrophoretic separation techniques in combination with sensitive element-specific detection methods. One unique property of all MT isoforms, besides their high metal binding capacity, is the high sulfur content found in these protein groups. Sulfur is the characteristic element found in the amino acids cysteine and methionine which occur 20 times and once, respectively, in a normal MT isoform consisting of 61 amino acids. Therefore the sulfur stoichiometry in different MT is constant whereas the composition of the bound trace elements may be different depending on the isoform, the state of the organism investigated, and its role in the whole metabolism. The known sulfur content offers the possibility to characterise the stoichiometric composition of MT isoforms and to quantify them.
Techniques such as flame photometry , total X-ray fluorescence spectrometry (TXRF)  and inductively coupled plasma (ICP) based detection techniques such as inductively coupled plasma–optical emission spectroscopy (ICP–OES) [8, 9] or inductively coupled plasma mass spectroscopy (ICP–MS) have been used for the determination of sulfur in different kinds of sample.
ICP–MS is one of the most sensitive methods for the simultaneous determination of trace elements in different matrices. However, one of the main drawbacks of ICP–MS is the formation of polyatomic ions in the 7000 K argon plasma generally used. Due to interferences caused by polyatomic ions at the mass of 32S (16O16O+, 31.98983 amu; 14N18O+, 32.00223 amu; 15N16O1H+, 32.00285 amu), high resolution ICP–MS is required in order to resolve the interferences from the elemental peak in the mass spectrum. A medium resolution of about m/Δm=4000 is sufficient for resolving the mass of 32S (at exactly 31.97207 amu) from the interferences. Prohaska et al. used ICP–SFMS combined with a membrane desolvation system for precise sulfur isotope ratio measurements in environmental samples . In combination with HPLC, ICP–SFMS was used for the determination of sulfur/phosphorus ratios in protein samples or for the sulfur-specific detection of impurities in sulfur-containing drugs [11, 12].
Quadrupole mass analyser-based ICP–MS systems have also been used for sulfur determination, either under normal or hyphenated conditions. Therefore the ICP–MS was tuned to generate high oxide ratios. Under more or less reproducible conditions, sulfur was measured as 32S16O+ using the corresponding m/z value of 48 for which interference is less [13, 14].
A new approach to the determination of non-metals with ICP mass spectrometry involves the use of collision or reaction cells in combination with less expensive quadrupole mass spectrometers in order to reduce interferences via chemical reactions or interactions with collision gases.
In general, two different approaches to the chemical resolution of interferences can be applied in the determination of 32S. The first approach is based on the chemical reaction of the analyte masses with reactive gases in order to create product ions which interfere less than the original masses. The use of gases such as N2O, CO2 or O2 for oxidation, or CH4 and C2H4 for methylation has been described. For the analysis of 32S, oxygen was successfully used to convert sulfur ions via an exothermal reaction into the masses of 32S16O+ (m/z 48) which interfere less . The second approach is based on the direct measurement of the most abundant analyte mass of 32S after removal of the interfering ions by using gases such as H2, He or Xe.
A number of workers have demonstrated the use of the isotope dilution technique for the quantification and characterisation of different metallothionein isoforms [18, 19] using capillary zone electrophoresis hyphenated with ICP–SFMS.
This paper will focus on the optimisation of an octopole reaction cell ICP–MS hyphenated with capillary electrophoresis for application to the separation and simultaneous element-specific determination of sulfur, copper, zinc, and cadmium in commercial MT preparations and in metallothionein-like proteins (MLP) extracted from bream liver samples, using Xe as a collision gas for minimisation of the interferences at the mass of 32S.
Chemicals and materials
Ultra-pure water (18 MOhm cm−1) was obtained from an Elix 3/Milli-Q Element system (Millipore, Milford, MA, USA). Nitric acid (Suprapur, 65%) and hydrochloric acid (Suprapur, 30%) were both obtained from Merck (Darmstadt, Germany). Both were cleaned by sub-boiling distillation in a quartz apparatus (Kürner, Rosenheim, Germany) and diluted with ultra-pure water to the desired concentration level. Ammonia solutions were prepared by dilution of 25% ammonia solution (Suprapur, Merck) with ultra-pure water. Potassium hydroxide solutions (1 and 0.1 mol L−1 KOH) were prepared by dissolving KOH·H2O (Suprapur, Merck) in ultra-pure water. Tris buffer solution was prepared by dissolving Tris(hydroxymethyl)aminomethane (p.a., Merck) in ultra-pure water and adjusting the solution with nitric acid to the desired pH. Hydrogen (H2, 5.0, 99.999%), helium (He, 5.0, 99.999%) and Xenon (Xe, 4.8, 99.998%) were purchased from Messer Griesheim (Krefeld, Germany). Rabbit liver metallothionein (MT, Lot 19H7812) and rabbit liver MT-1 (Sigma MT-1, Lot 127H7810) were purchased from Sigma (Deisenhofen, Germany). A single-element standard of S (1000 mg L−1) and a multi-element standard (Merck IV) were both Certipur for ICP–MS and purchased from Merck. Cadmium nitrate tetrahydrate (p.a.) was also purchased from Merck. Thiourea was purchased from Sigma (Sigma, Deisenhofen, Germany). Chelex 100 ion-exchange resin (Sigma) was used for buffer purification. Ultra filtration units (Viva Spin 100 kDa MWCO PES) were purchased from Satorius Viva Science (Göttingen, Germany). Units with a MWCO of 3 kDa were purchased from Microcon (Microcon Millipore, Bedford, MA, USA).
The instrumental set-up for all CE–ICP–MS experiments consists of an Agilent 3D capillary electrophoresis system (Agilent Technologies, Waldbronn, Germany) and an Agilent 7500c quadrupole ICP–MS with a shield-torch system and an octopole reaction system (Agilent Technologies, Tokyo, Japan). Both instruments were coupled via an interface based on a modified micro-concentric nebulizer (CEI 100, CETAC Technologies, Omaha, Nebraska, USA). Details of the interface construction can be found in previous publications [20, 21]. The CE–ICP–MS interface was adjusted to give a continuous flow rate of about 10 μL min−1 in order to minimise the intake into the plasma. A 10 mmol L−1 ammonia solution adjusted to pH 7.4 with nitric acid was used as make-up liquid. A spike of 40 μg Ge L−1 was used as internal standard and for monitoring the stability of nebulization during CE experiments. The ICP–MS was linked to the interface by a shielded Teflon tube connected directly with the torch.
Capillary conditioning parameters for CE–ORS–ICP–MS
Concentration (mol L–1)
Flush capillary with 100 kPa
Flush capillary with 100 kPa
Flush capillary with 100 kPa
Flush capillary with 100 kPa
Flush capillary with 100 kPa
The auto-sampler of the CE was kept at a constant temperature level of +10 °C with an external cooling system to prevent changes in the viscosity of the sample and the running buffer.
Cetac CEI 100
PFA Micro flow 100
RF power (W)
Extraction lens (V)
+4 to +6
+4 to +6
Sampling depth (mm)
Carrier gas (L min–1)
Make-up gas (L min–1)
OctP bias (V)
QP bias (V)
Xe flow (%)
Capillary length (cm)
Capillary i.d. (mm)
Separation voltage (kV)
Vial table temperature
Hydrodynamically with different volumes
Tris-HNO3 20 mmol L–1, pH 7.0–7.4
MT solutions of different concentration were prepared gravimetrically by dissolving appropriate amounts of MT in degassed, argon-flushed buffer solution. MT solutions were stored under argon at –18 °C until analysis.
The Elbe bream investigated in this work were obtained from local fishermen working in the region of Gorleben, Lower Saxony, Germany. The fish were killed directly after being caught, washed with tap water and stored in polyethylene (PE) plastic bags at –18° C until further preparation.
Cytosol preparation/extraction of the metallothionein-like proteins
For cytosol preparation about 3 g of liver tissue was transferred into a Potter–Elvehjem homogenisation tube together with 3 mL of cooled (+4 °C), degassed, argon-flushed, Tris-HNO3 buffer (5 mmol L−1, pH 7.4). All sample preparation steps were performed with ice cooling and in an argon atmosphere to prevent oxidation of the sample. The use of reducing agents such as 2-mercaptoethanol or dithiothreitrol was avoided due to their metal-complexing capabilities which could influence the metal composition of the species investigated.
In order to avoid contamination, especially with the elements copper and zinc, all tools (vials, caps, containers bottles etc.) used in sample preparation were cleaned with acid before use.
The liver tissue was homogenised within a few minutes. The homogenate was then centrifuged at 25,000 g for 90 min at +4 °C. The supernatant was separated from the tissue residues and stored under argon at –18 °C until required.
For further sample purification, ultra filtration units with a molecular weight cut-off (MWCO) of 100,000 Da were used to remove high-molecular-weight proteins and particles from the cytosol. Therefore the supernatant was transferred under argon atmosphere into a filtration unit and centrifuged at +4 °C and 8000 g. For the sulfur experiments, pre-concentration of the cytosol was performed. The filtrate obtained in the previous preparation step (2 mL) was transferred into another filtration unit with a MWCO of 3000 Da and centrifuged at 8000 g until a final volume of about 200 μL was achieved.
All element standards were prepared by dilution of the stock solution with ultra-pure water to the desired concentration. The solutions were stored in PFA or FEP containers cleaned with acid vapour to minimise contamination. All standards were measured directly after preparation.
Results and discussion
Optimisation of 32S detection
The instrument was tuned without using the collision/reaction mode on a daily basis with a standard tuning solution (1 μg Li, Y, Ce, Tl) to obtain maximum sensitivity and to optimise the nebulization efficiency of the CE–ICP–MS interface.
Abundances of sulfur isotopes and relevant polyatomic ions
The grey spectrum demonstrates the effect of addition of Xe (5%, approx. 0.2 mL min−1) to the octopole reaction system on the spectral background of the same DI water sample. The spectral background is decreased by about six orders of magnitude enabling the determination of sulfur with quadrupole-based ICP–MS using the most abundant isotope 32S.
Instrumental detection limits
Detection limits estimated according to DIN 32645 for sulfur and some simultaneously measured trace elements in an aqueous solution (MilliQ DI water) measured in different ICP–MS modes
Detection limits Xe mode (μg L–1)
Detection limits standard mode (ng L–1)
Detection limits H2 mode (ng L–1)
Detection limits He mode (ng L–1)
Analysis of an MT preparation
Quantification of sulfur and cadmium in an MT-1 preparation using external calibration
Detection limits estimated according to DIN 32645 for sulfur and cadmium obtained under CE–ORS–ICP–MS conditions
Detection limits (mg L–1)
Absolute detection limits (pg)
Quantification of the sulfur and cadmium content of a commercial MT-1 preparation. Concentrations given in mg L–1
Element-specific determination of metallothionein-like proteins in liver cytosol samples of Abramis brama L. using CE–ORS–ICP–MS
The investigation of real-world samples containing metallothioneins at natural levels represents a challenging task in metallothionein speciation with CE–ORS–ICP–MS. The main problems are related to the low MT concentration levels found in real samples and the poor detection limits achieved with CE–ICP–MS due to the small sample amounts generally used in CE experiments (normally a few nL). A further problem results in the reduced ion transmission when using Xe as collision gas for sulfur measurement. Therefore, sample clean-up and pre-concentration steps are necessary in order to obtain an adequate sample concentration. Ultra-filtration was used to minimise the interaction of the sample with other chemicals and to avoid the formation of artefacts during sample preparation.
ORS–ICP–MS is a suitable technique for the simultaneous determination of sulfur and other trace elements either under normal ICP–MS or under hyphenated conditions. Xenon is an effective collision gas for reducing interference at the masses of sulfur. CE–ORS–ICP–MS allows the simultaneous detection of sulfur, cadmium, copper, and zinc in biologically relevant molecules such as metallothionein or metallothionein-like proteins. External calibration based on the use of sample independent, element-containing compounds is an appropriate technique for the quantification of sulfur and cadmium in an MT-1 preparation. For quantification of real samples a further improvement of the sample-preparation technique is necessary. Comparable results were obtained with TXRF as an independent technique for total element quantification. Further experiments will be necessary to apply the same technique for the quantification of copper and zinc. A promising approach is the use of 2,6-diacetylpyridine-bis(N-methylenepyridiniohydrazone) as a complexing agent for copper and zinc. This enables stable, positively charged complexes under the pH conditions usually used for MT separation to be generated. Finally, CE–ORS–ICP–MS offers enough sensitivity for the separation and element-specific detection of metallothionein-like proteins at a non-induced level in bream liver samples via measurement of their sulfur, copper, zinc and cadmium contents.
The authors would like to thank Simone Griesel and Ulrich Reus for performing the TXRF measurements.
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