Speciation of trace elements in proteins in human and bovine serum by size exclusion chromatography and inductively coupled plasma-mass spectrometry with a magnetic sector mass spectrometer

 Proteins are separated by size exclusion chromatography while atomic ions from the inorganic elements are detected on-line by inductively coupled plasma-mass spectrometry. A double focusing mass analyzer provides very high sensitivity, low background, and sufficient spectral resolution to separate the atomic ions of interest from most polyatomic ions at the same nominal m/z value. The chromatograms show the distribution of the elements of interest between protein-bound and free fractions and provide the approximate molecular weights of those protein fractions that contain the elements monitored. The distribution of various elements, including V, Mo, Fe, Co, Mn, and lanthanides, in human or bovine serum samples are shown. Alkali metals and Tl are present primarily as free metal ions and are not bound to proteins. Inorganic elements spiked into the serum samples can be followed into various proteins. EDTA does not remove Fe, Pb, Sn, or Th from the proteins but does extract Mn from some proteins. Procedures for determining the effects of breaking disulfide linkages on the metal binding characteristics of proteins are also described. Received: 22 March 1999 / Accepted: 16 June 1999     

Fractionation of phosphorus and trace elements species in soybean flour and common white bean seeds by size exclusion chromatography-inductively coupled plasma mass spectrometry

Soluble species of phosphorus, sulfur, selenium and eight metals (Mn, Fe, Co, Ni, Cu, Zn, Mo and Cd) in soybean flour and common white bean seeds were investigated by size exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry (ICP-MS). Samples were extracted by 0.02 mol l(-1) Tris-HCI buffer solution (pH 7.5). Fractionation of sample extracts by preparative scale SEC was accomplished using a Fractogel EMD BioSEC column (600 x 16 mm) and 0.02 mol l(-1) Tris-HCl buffer solution (pH 7.5) as mobile phase (flow rate: 2 ml min(-1)). A 2-ml sample was injected. Contents of elements in chromatographic fractions were determined by AAS, ICP-AES and ICP-MS. The elution profiles of P, Fe, Co, Ni, Cu, Zn and Mo in both samples were similar. Main species of Co, Ni, Cu, Zn and Mo were found in the low molecular weight region (2-5 kDa), whereas Fe is predominantly bound to high molecular weight compounds (180 kDa). The dominant phosphorus fraction was detected in the medium molecular weight region (10-30 kDa) and the other fraction in the low molecular weight region. Isotachophoretic analysis of chromatographic fractions revealed that the main phosphorus compound in the medium molecular weight region is phytic acid. SEC on Superdex 75 and Superdex Peptide columns (300 x 10 mm) was performed in on-line hyphenation with ICP-MS. The same mobile phase was used with a flow rate of 0.5 ml min(-1); volume of injected sample was 200 microl. Element specific chromatograms were obtained by continuous nebulization of effluent into ICP-mass spectrometer measuring intensities of 47(PO)+ and 48(SO)+ oxide ions and 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se, 95Mo and 114Cd nuclides. Chromatographic profiles of elements are generally analogous to those obtained with a Fractogel column, but better chromatographic resolution of separated species was achieved so that slight differences between samples were revealed. Estimated molecular weights of major phosphorus species in soybean flour and common white bean seed extracts are 6 and 3.6 kDa, respectively, whereas those of minor phosphorus species in both samples are 0.7 kDa. Traces of phosphorus were also detected in the high molecular weight region (130 kDa). Chromatograms of P, Ni, Cu, Zn and Mo compounds in both extracts are similar but not identical. Molecular weights of major Cu and Zn species are approximately 1 and 0.4 kDa for soybean flour and white bean seeds, respectively. In cases of Mn, Fe, Co and Se, the element profiles of soybean flour and white bean seed extracts are significantly different.     

Concentrations of selected trace elements in human milk and in infant formulas determined by magnetic sector field inductively coupled plasma-mass spectrometry

Magnetic sector field inductively coupled plasma-mass spectrometry (ICP-MS) was applied to the reliable determination of the 8 essential trace elements cobalt (Co), chromium (Cr), copper (Cu), iron (Fe), manganese (Mn), nickel (Ni), selenium (Se), and vanadium (V) as well as the 7 nonessential and toxic elements silver (Ag), aluminum (Al), arsenic (As), gold (Au), platinum (Pt), scandium (Sc), and titanum (Ti) in 27 transitory and mature human milk samples and in 4 selected infant formulas. This advanced instrumentation can separate spectral overlaps from the analyte signal hampering significantly the determination of many trace elements by conventional ICP-MS. Moreover, superior detection limits in the picogram per liter range can be obtained with such magnetic sector field instruments. Therefore, this is the first study to report the concentrations of the elements Ag, Au, Pt, Sc, Ti, and V in human milk and in infant formulas. Concentrations of Ag (median: 0.41 μg/L; range: <0.13–42 μg/L) and Au (median: 0.29 μg/L; range 0.10–2.06 μg/L) showed large variations in human milk that might be associated with dental fillings and jewelry. Pt concentrations were very low with most of the samples below the method detection limit of 0.01 μg/L. Human milk concentrations of Co (median: 0.19 μg/L), Fe (380 μg/L), Mn (6.3 μg/L), Ni (0.79 μg/L), and Se (17 μg/L) were at the low end of the corresponding reference ranges. Concentrations of Cr (24.3 μg/L) in human milk were five times higher than the high end of the reference range. For Al (67 μg/L), As (6.7 μg/L), and V (0.18 μg/L), most of the samples had concentrations well within the reference ranges. All elemental concentrations in infant formulas (except for Cr) were approximately one order of magnitude higher than in human milk.     

Multianalysis of trace elements in mosses with inductively coupled plasma-mass spectrometry

As a part of an air-pollution biomonitoring survey, a procedure using inductively coupled plasma-mass spectrometry (ICP-MS) and microwave digestion was developed to achieve a high sample throughput and guarantee the accuracy of the results. This article presents an analytical method to measure 22 trace elements. As, Ba, Cd, Ce, Co, Cr, Cs, Cu, Fe, Hg, La, Mo, Ni, Pb, Rb, Sb, Sr, Th, Tl, U, V, W were analyzed in 563 mosses collected in France. The digestion was performed in polytetrafluoroethylene (PTFE) vessel using the mixture HNO₃-H₂O₂-HF. The data were reprocessed taking into account the drift curve calculated for each element. The detection limits (DL) calculation was based on the standard deviations of the reagent blanks concentrations. The DL varied from one batch to another, because of the heterogeneity of the mosses’ elemental contents. The DL ranged between 0.001 μg/g (Cs, Tl) and 70 μg/g (Fe) and were mainly around 0.01 μg/g (As, Cd, Ce, Co, Hg, La, Mo, Sb, Sr, U, V, W). The detection limits obtained were in agreement with the concentrations observed in the samples, except for Hg and Ni. The reproducibility between duplicates and the analytical precision were near 10%. The procedure was tested with the mosses’ reference materials.     

Characterisation of zinc-binding domains of peroxisomal RING finger proteins using size exclusion chromatography/inductively coupled plasma-mass spectrometry

We determined the zinc binding stoichiometry of peroxisomal RING finger proteins by measuring sulfur/metal ratios using inductively coupled plasma-mass spectrometry coupled to size exclusion chromatography, a strategy that provides a fast and quantitative overview on the binding of metals in proteins. As a quality control, liquid chromatography-electrospray ionisation-time of flight-mass spectrometry was used to measure the molar masses of the intact proteins. The RING fingers of Pex2p, Pex10p, and Pex12p showed a stoichiometry of 2.0, 2.1, and 1.2 mol zinc/mol protein, respectively. Thus, Pex2p and Pex10p possess a typical RING domain with two coordinated zinc atoms, whereas that of Pex12p coordinates only a single zinc atom.     

Determination of major and trace elements in the liver of Wistar rats by inductively coupled plasma-atomic emission spectrometry and mass spectrometry

Inductively coupled plasma-mass spectrometry (ICP-MS) and inductively coupled plasma-atomic emission spectrometry (ICP-AES) were used to determine age-related changes in the concentrations of constituent elements in the livers of Wistar rats of 1 week to 12 months old. At first, sample preparation and analytical conditions were investigated in order to set up a simple routine procedure for measuring multiple elements simultaneously. Seventeen elements in the standard reference samples of bovine and pork livers as well as rat liver samples could be determined with a reasonable precision and reproducibility. They were P, K, Na, Fe, Mg, Ca, Zn, Rb, Cu, Mn, Mo, Al, Co, Sr, Cs, Pb, and Cd in order of the levels of concentration in the adult rat livers. Of these elements, the five major elements (P, K, Na, Fe, Mg, Ca) were determined with ICP-AES and the others with ICP-MS. Although the number of animals was too small to draw a statistically definite conclusion, it seems that age-related changes in the concentrations of these elements could be categorized into three general patterns: (1) remaining essentially constant throughout the animal ages, as observed for P, K, Na, Mg, Ca, Rb, Sr, Cs, and Pb, (2) increasing with age, as observed for Fe, Mn, Mo, Co, and Cd, and (3) decreasing with age, especially in the early stages of growth, as observed for Cu and Zn.     

Speciation of trace elements in human serum by micro anion exchange chromatography coupled with inductively coupled plasma mass spectrometry

Speciation analysis of essential trace elements in human serum provides important information on nutritional status and homeostatic mechanisms regulating transport processes, acute phase reactions, and protection against oxidative damage. Anion exchange high-performance liquid chromatography (HPLC) combined with inductively coupled plasma mass spectrometry (ICP-MS) has proved to be a useful tool in speciation. Here we describe a fast method that can be applied to carry out the speciation of Fe, Cu, Zn, and Se in as little as 1 microl [corrected] of serum. The method employs monolithic anion exchange micro columns installed on a tandem HPLC system coupled on-line with an ICP-MS detector. The chromatographic separation is similar to those reported previously but with considerable gain in terms of time and sample requirement. Reproducibility is acceptable for most species. Using our method, we were able to find species-specific differences between different commercially available trace element reference materials. Because the method chosen to collect blood might interfere with speciation, the proposed methodology was used to compare heparinized plasma, ethylenediaminetetraacetic acid (EDTA) plasma, and serum from adult healthy volunteers. As expected, EDTA strongly affects speciation analysis (especially for Fe and Zn), whereas changes due to the use of lithium-heparin (Li-He) as anticoagulant appear to be minimized.     

Determination of trace elements in porcine brain by inductively coupled plasma-mass spectrometry,electrothermal atomic absorption spectrometry,and instrumental neutron activation analysis

Methods have been developed for the analyses of trace metals in various areas of porcine brains, (temporal, parietal, frontal cortex, both right and left hemispheres). Determinations were carried out using inductively coupled plasma-mass spectrometry (ICP-MS) and electrothermal atomic absorption spectrometry (ETAAS). The elements investigated were Li, Mn, Cu, Zn, Cd, Hg, and Pb by ICP-MS and Cu, Cd, and Mn by ETAAS. For determination by ICP-MS, a method of standard additions calibration coupled with internal standards was used, and for ETAAS, standard additions calibrations were prepared. The accuracy of all methods was determined using NIST and IAEA certified reference material. A small number of pig brains were analyzed by instrumental neutron activation analysis for Cr, Co, Cs, Fe, Rb, Se, Sc, Sb, and Zn using the comparator method of analysis. Four separate NIST standard reference materials have been used to examine the validity of the comparator method.     

Preparation of selenium-enriched sprouts and identification of their selenium species by high-performance liquid chromatography-inductively coupled plasma mass spectrometry

Sprouts of several plants (10 families and 28 species) were cultivated in a high selenium environment, and the chemical species of selenium in these selenium-enriched sprouts were identified by using high-performance liquid chromatography-inductively coupled plasma mass spectrometry (HPLC-ICPMS). Cultivation of sprouts of kaiware daikon (type of radish) with 5.0 microg/ml or 10.0 microg/ml of selenium as selenite inhibited the growth. However, no abnormalities in the shape or color were apparent even in the sprouts exposed to 10.0 microg/ml of selenium. The selenium concentration in the sprouts of most plants examined was higher than that from environmental exposure. Among the types of selenium that were accumulated, a large part (69-98%) was extractable in 0.2 M HCl. Chemical analysis of selenium in the HCl extract showed that the main selenium species in all the sprouts examined was Se-methylselenocysteine. In addition to Se-methylselenocysteine, selenomethionine, non-metabolized selenite, gamma-glutamyl-Se-methylselenocysteine and an unknown selenium compound were also detected in several high-selenium sprouts. Since higher anticarcinogenic activities of these monomethylated selenoamino acids have been observed, it is anticipated that such selenium-enriched sprouts will be used as a foodstuff for cancer prevention.     

Determination of trivalent methylated arsenicals in rat urine by liquid chromatography-inductively coupled plasma mass spectrometry after solvent extraction

A method for the determination of trivalent arsenicals in urine was examined. Trivalent arsenicals, extracted as complexes with diethylammonium diethyldithiocarbamate (DDDC) into carbon tetrachloride, were determined by liquid chromatography-inductively coupled plasma mass spectrometry (LC-ICP-MS). The trivalent methylated arsenicals monomethylarsonous acid (MMA(III)), dimethylarsinous acid (DMA(III)), and trimethylarsine (TMA) were detected in urine of rats that had received dimethylarsinic acid (DMA(V)) or monomethylarsonic acid (MMA(V)) at concentration of 200 microg ml(-1) in drinking water for 24 weeks. This method is the first to permit quantification of trivalent methylated arsenicals in urine without significant changes in concentration during storage or pretreatment.     

Application of size-exclusion chromatography-inductively coupled plasma mass spectrometry for fractionation of element species in seeds of legumes

Fractionation of soluble species of P, Mn, Fe, Co, Ni, Cu, Zn, Se and Mo in pea and lentil seeds was made by on-line hyphenation of size-exclusion chromatography (SEC) and inductively coupled plasma mass spectrometry. Seed samples were extracted with 0.02 mol l(-1) Tris-HCl buffer solution, pH 7.5. SEC was performed on Superdex 75 and Superdex Peptide columns (300 x 10 mm) with the same buffer solution as the mobile phase. Monitoring of oxide ion 47(PO)+ was used for detection of phosphorus compounds. Other elements were detected as ions of 55Mn, 57Fe, 59Co, 62Ni, 65Cu, 66Zn, 82Se and 95Mo nuclides. Elements in individual elution zones were quantified using external calibration. Complete chromatographic recoveries of elements were found in cases of phosphorus, nickel and copper. Substantial parts of manganese and zinc, as well as traces of cobalt, selenium and molybdenum are retained on the column. Injection of EDTA solution removes these elements from the column. Chromatographic profiles of pea and lentil samples are very similar for all elements except Mo. Main element species in the high-molecular-mass region (approx. 190,000 rel. mol. mass unit) were detected in case of Fe. Low-molecular-mass species (<2000 rel. mol. mass unit) as major element forms are typical for Cu and Zn.     

Measurement of ergotamine in human plasma by triple sector quadrupole mass spectrometry with negative ion chemical ionization

By use of negative ion chemical ionization and collision-activated decomposition in a triple quadrupole mass spectrometer a method has been developed for the quantification of ergotamine in human plasma at levels down to 2 pg ml-1.     

Characterization of metallothionein isoforms from rabbit liver by liquid chromatography coupled to electrospray mass spectrometry

Metallothioneins (MTs), a group of low molecular weight proteins found in practically all life forms, are characterized by high sulfur content and an affinity for metal ions. At acidic pH, MTs show metal depletion, leading to apothioneins. In the work described here, in order to optimize the separation of rabbit liver apothioneins using liquid chromatography (LC) with UV detection, the proportion of the organic modifier of the mobile phase was optimized by establishing relationships between Reichardt's E(N)(T) scale of solvent polarity and the chromatographic retention measured by the capacity factor, k. Additionally, such optimum separations were carried out in a LC-electrospray mass spectrometry (ES-MS) coupled system allowing the identification and characterization of the different rabbit liver apo-MT-forms. In this way, electrospray ionization mass spectrometry offers great possibilities aiming at a better understanding of metallothionein polymorphism.     

Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography

Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.     

Speciation of tissue and cellular iron with on-line detection by inductively coupled plasma-mass spectrometry.

Iron accumulating to excess in tissues of humans and animal models occurs mainly as complexes with transferrin, ferritin, other hemoproteins, and insoluble hemosiderin particles. To determine the distribution of Fe amongst these molecular species, we have used inductively coupled plasma-mass spectrometry as a means of on-line, isotope-specific detection for their liquid chromatographic separation. The stable isotope 57Fe is a suitable isotope for monitoring the Fe content of each fraction, and its availability at high isotopic enrichment makes it an attractive choice for tracer studies when the use of a radioisotope is undesirable, e.g., in human subjects. The detection system offers the advantages of high sensitivity (detection limits in the parts per billion range), a wide dynamic range (linearity of the calibration curve over several orders of magnitude), and on-line analysis facilitating real-time evaluation of the chromatographic separation, in addition to isotope-specific information. The Fe distributions in healthy rat livers, liver and heart tissue from Fe-loaded human subjects, and human hepatocyte cultures are reported. The ferritin:hemosiderin ratio in these samples is shown to be an indicator of the degree of Fe loading and correlates well with that determined by Zeeman-corrected electrothermal atomic absorption as an alternative means of detection.     

Composition of the major elements and trace elements of 10 methanogenic bacteria determined by inductively coupled plasma emission spectrometry

The elemental composition of 10 methanogenic species was determined by inductively coupled plasma emission spectrometry and by a C-H-N-analyzer. The 10 species were representative of all three orders of the methanogens and were cultivated under defined conditions. Special emphasis was given toMethanosarcina barkeri, represented by 5 strains and cultivated on various substrates. The following elements with the lowest and highest values in parentheses were determined: C (37–44%, w/w), H (5.5–6.5%), N (9.5–12,8%); Na (0.3–4.0%), K (0.13–5.0%), S (0.56–1.2%), P (0.5–2.8%), Ca (order I: 85–550 ppm; order II: 1000–4500 ppm), Mg (0.09–0.53%), Fe (0.07–0.28%), Ni (65–180 ppm), Co (10–120 ppm). Mo (10–70 ppm), Zn (50–630 ppm), Cu (<10–160 ppm), Mn (<5–25 ppm). The biggest variations were found with respect to N and K, which both seem to have important physiological functions. Although it is unknown whether zinc and copper are essential trace elements for methanogens, all investigated species contained remarkably high zinc contents, whereas copper seemed to be present only in some species.     

Detection and characterization of zinc- and cadmium-binding proteins in Escherichia coli by gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry

Metals bound to proteins play key roles in structure stabilization, catalysis, and metal transport in cells, but metals may also be toxic. As a consequence, cells have developed mechanisms to control metal concentrations through binding to proteins. We have used a hyphenated strategy linking gel electrophoresis with laser ablation-inductively coupled plasma-mass spectrometry in order to detect, map, and quantify metal-binding proteins synthesized in Escherichia coli under zinc- and cadmium-stress conditions. We report the development of a powerful analytical method suitable for detection and characterization of metalloproteins in complex, unfractionated bacterial cell extracts. The approach was validated by using an E. coli strain overexpressing the cyanobacterial metallothionein protein SmtA. We observed induction of SmtA synthesis by zinc and binding of both zinc and cadmium cations by this protein. A profile of zinc- and cadmium-binding proteins was obtained from E. coli cytoplasmic fractions. Analysis of induction patterns and metal contents demonstrated the presence of proteins with high metal content which, on further study, should lead to the identification of novel metal-binding proteins.     

Accurate mass measurement using multiple sprayer nano-electrospray mass spectrometry combined with nano-scale high-performance liquid chromatography on a magnetic sector instrument

A new technique for accurate mass measurement utilizing multiple sprayer nano-electrospray ionization mass spectrometry (nano-ESI-MS) combined with nano-scale high-performance liquid chromatography (nano-HPLC) on a magnetic sector instrument is described. Both metal-coated glass capillaries and fused-silica capillaries were used as nano-ESI sprayers. A metal-coated glass capillary was used for the introduction of the Ref. compound solution, and a metal-coated fused-silica capillary was used for connection to the nano-HPLC column. By shifting each sprayer's position relative to the sampling orifice, spectra were obtained of both the sample components as eluted from the column and reference compounds. Several standard compounds were examined and satisfactory accurate masses were obtained. Problems arising from differences in ionization efficiency between the sample and reference compounds were not observed.     

Trace level determination of trichloroethylene from liver,lung and kidney tissues by gas chromatography-magnetic sector mass spectrometry

Trichloroethylene (TCE) is a common industrial chemical that has been heavily used as a metal degreaser and a solvent for the past 100 years. As a result of the extensive use and production of this compound, it has become prevalent in the environment, appearing at over 50% of the hazardous waste sites on the US EPA's National Priorities List (NPL). TCE exposure has been linked to neurological dysfunction as well as to several types of cancer in animals. This paper describes the development and validation of a gas chromatography-mass spectrometry (GC-MS) method for the quantitation of trace levels of TCE in its target tissues (i.e. liver, kidney and lungs). The limit of quantitation (5 ng/ml) is substantially lower than currently published methods for the analysis of TCE in tissues. The % RSD and % Error for the assay falls within the acceptable range (<15% for middle and high QC points and <20% for low QC points), and the recovery is high from all tissues (>79%).