Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.     

Inorganic trace analysis by mass spectrometry

Mass spectrometric methods for the trace analysis of inorganic materials with their ability to provide a very sensitive multielemental analysis have been established for the determination of trace and ultratrace elements in high-purity materials (metals, semiconductors and insulators), in different technical samples (e.g. alloys, pure chemicals, ceramics, thin films, ion-implanted semiconductors), in environmental samples (waters, soils, biological and medical materials) and geological samples. Whereas such techniques as spark source mass spectrometry (SSMS), laser ionization mass spectrometry (LIMS), laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS), glow discharge mass spectrometry (GDMS), secondary ion mass spectrometry (SIMS) and inductively coupled plasma mass spectrometry (ICP-MS) have multielemental capability, other methods such as thermal ionization mass spectrometry (TIMS), accelerator mass spectrometry (AMS) and resonance ionization mass spectrometry (RIMS) have been used for sensitive mono- or oligoelemental ultratrace analysis (and precise determination of isotopic ratios) in solid samples. The limits of detection for chemical elements using these mass spectrometric techniques are in the low ng g⁻¹ concentration range. The quantification of the analytical results of mass spectrometric methods is sometimes difficult due to a lack of matrix-fitted multielement standard reference materials (SRMs) for many solid samples. Therefore, owing to the simple quantification procedure of the aqueous solution, inductively coupled plasma mass spectrometry (ICP-MS) is being increasingly used for the characterization of solid samples after sample dissolution. ICP-MS is often combined with special sample introduction equipment (e.g. flow injection, hydride generation, high performance liquid chromatography (HPLC) or electrothermal vaporization) or an off-line matrix separation and enrichment of trace impurities (especially for characterization of high-purity materials and environmental samples) is used in order to improve the detection limits of trace elements. Furthermore, the determination of chemical elements in the trace and ultratrace concentration range is often difficult and can be disturbed through mass interferences of analyte ions by molecular ions at the same nominal mass. By applying double-focusing sector field mass spectrometry at the required mass resolution—by the mass spectrometric separation of molecular ions from the analyte ions—it is often possible to overcome these interference problems. Commercial instrumental equipment, the capability (detection limits, accuracy, precision) and the analytical application fields of mass spectrometric methods for the determination of trace and ultratrace elements and for surface analysis are discussed.     

Verbundverfahren zur Multielement-Spurenbestimmung in hochreinem Chrom nach Spuren—Matrix-Trennung

(Simultaneous determination of trace elements in high-purity chromium by inductively-coupled plasma/atomic emission spectrometry after matrix separation.) The production of high-purity metals requires routine determinations of elements in the ng g^?1 range. Procedures based on wet chemical separation of matrix and trace elements followed by inductively-coupled plasma/atomic emission spectrometry are suitable. The separation of 19 trace metals (Be, Bi, Ca, Cd, Co, Cu, Fe, La, Mg, Mn, Nb, Ni, Pb, Ta, Ti, Th, U, Zn and Zr) from high-purity chromium powder is described. The powder is dissolved in hydrochloric acid and oxidized with perchloric acid or alkaline hydrogen peroxide, and the trace elements are precipitated at pH 11–13 and collected on cellulose loaded with indium hydroxide or on cellulose-Hyphan. The detection limits of the total procedure vary from 10 ng g^?1 for cadmium to 600 ng g^?1 for zinc.     

金属蛋白的提取分离技术

微量元素在生命活动中起着十分重要的作用。体内微量元素(主要指金属元素)水平的反常、存在形态的变化以及特定的金属酶、金属蛋白的表达、定位、结构和功能等方面的变化常常与某些病理状态相关,甚至是关键的病理环节,所以对生物体系中金属蛋白的定位、含量、化学形态及其结构的高通量分析是生命科学研究领域的重要课题。本文介绍并比较了各种金属蛋白的提取、分离方法(包括高效液相、毛细管电泳和电泳技术)及与之对应的元素分析技术(包括激光剥蚀(LA)-等离子体电感耦合质谱(ICP-MS)、同步辐射(SR)-X射线荧光(XRF)技术等),主要介绍了各种电泳方法在金属蛋白分离中的应用。同时评价各种方法对蛋白结合金属的影响,针对方法的局限性提出了可能的解决办法。     

Characterization of biological macromolecules by electrophoresis and neutron activation

As analytical techniques have become more sensitive, the role of trace elements associated with biological macromolecules has become the subject of many studies in the past twenty years. Many biologically significant macromolecules, such as nucleic acids and proteins, have trace elements essential to their function and structure. Instrumental neutron activation analysis (INAA) contributes useful information in such studies. A procedure combining polyacrylamide gel electrophoresis (PAGE) with INAA and autoradiography has been developed to study biological macromolecules and their associated trace elements. Results from the application of this method to several metalloproteins are presented.     

Inductively Coupled Plasma(ICP) Mass Spectrometry(MS) Hyphenated with Atomic Emission Spectrometry(AES) for Simultaneous Determination of Major, Minor and Micro Amounts of Elements in Geochemical Samples

Introduction Geological resource survey demands for determining various constituents including major, minor, micro, trace and ultra-trace levels of elements for preparing the map of resource distribution of our country. As a powerful and popularly used technique for multi-element analysis, inductively coupled plasma(ICP) atomic emission spectrometry (AES) has been applied to this field for a period of time[1-3]. However, ICP spectrometric determination of those micro, trace and ultratrace elements needs enrichment procedures for improving the detection limit, which is unacceptable in case a great mass of samples should be analyzed as that in the task of geological resource survey. On the other hand, although ICP mass spectrometry(MS) is considered the most powerful method for trace elements determination[4,5], it is difficult for ICP-MS to be used to determine the trace and major analytes simultaneously in a spectrum.     

Evaluation of gel electrophoresis conditions for the separation of metal‐tagged proteins with subsequent laser ablation ICP‐MS detection

Although laser ablation (LA)‐ICP‐MS has been reported for the determination of metalloproteins separated by gel electrophoretic techniques (GE), systematic studies that define the conditions essential for successful measurements are still scarce. In this paper we present the results of our studies of basic conditions for the effective application of GE‐LA‐ICP‐MS for the separation of metal‐binding proteins, focusing on their stability during GE and post‐separation gel treatment. The stability of metal–protein complexes (haemoglobin, myoglobin, superoxide dismutase, carbonic anhydrase, transferrin, albumin, cytochrome c) during GE is dependent on the nature of the metal–protein interaction and the principle of separation. We have observed that non‐denaturing GE is a suitable separation technique for most metal–protein complexes (e.g. Zn in carbonic anhydrase and Fe in Tf and myoglobin were quantitatively recovered in a spiked liver cytosol), whereas separation by denaturing GE strongly impaired the stability of the complexes. Equally important is the post‐separation treatment of the gel to enable successful detection of the metal. LA‐ICP‐MS requires drying of the gel without loss of protein‐bound metal or cracking of the gel. This was successfully achieved using glycerol followed by heating. We demonstrate that staining of the gel prior to LA‐ICP‐MS using silver or Coomassie blue is not recommended, since most protein‐bound metal is lost during the staining procedure. Furthermore it has been shown that only line scanning with a speed of less than 30 μm/s can reliably distinguish between lines 1 mm apart, while raster spot analysis carries the risk of misinterpretation due to contamination in/on inhomogeneous gels.     

Evaluation of an ultrasonic nebulizer-membrane separation interface (USN-MEMSEP) with ICP-AES for the determination of trace elements by solvent extraction

The introduction of volatile organic solvents and metal organic complexes into an inductively coupled plasma (ICP) is problematic due to overloading and pyrolysis effects. These include carbon built up in the torch and spectral interferences. As a consequence, solvent extraction as a method for preconcentrating trace metals for the determination by ICP has been limited. In this report a commercial ultrasonic nebulizer-membrane separation interface (USN-MEMSEP) for the direct introduction and separation of organic solvents using ICP atomic emission spectrometry (AES) and a sequential spectrometer has been evaluated for solvent extraction of chelated trace metals. The ability of the MEMSEP to separate volatile organic flows from metal aerosols has been demonstrated by determining the recoveries of several transition metals in an oil-based methyl-isobutyl ketone (MIBK) standard relative to an aqueous solution. However, low recoveries of several metal chelates have been found evidently due to the volatilization of the organic metal species at the boiling point of MIBK (160° C). Moreover, the multielement capability and limits of detection have been limited due to sequential atomic emission detection. Advantages of the technique include enhanced limits of detection (LODs) and reduced plasma and spectral interferences.     

Evaluation of an ultrasonic nebulizer-membrane separation interface (USN-MEMSEP) with ICP-AES for the determination of trace elements by solvent extraction

The introduction of volatile organic solvents and metal organic complexes into an inductively coupled plasma (ICP) is problematic due to overloading and pyrolysis effects. These include carbon built up in the torch and spectral interferences. As a consequence, solvent extraction as a method for preconcentrating trace metals for the determination by ICP has been limited. In this report a commercial ultrasonic nebulizer-membrane separation interface (USN-MEMSEP) for the direct introduction and separation of organic solvents using ICP atomic emission spectrometry (AES) and a sequential spectrometer has been evaluated for solvent extraction of chelated trace metals. The ability of the MEMSEP to separate volatile organic flows from metal aerosols has been demonstrated by determining the recoveries of several transition metals in an oil-based methyl-isobutyl ketone (MIBK) standard relative to an aqueous solution. However, low recoveries of several metal chelates have been found evidently due to the volatilization of the organic metal species at the boiling point of MIBK (160° C). Moreover, the multielement capability and limits of detection have been limited due to sequential atomic emission detection. Advantages of the technique include enhanced limits of detection (LODs) and reduced plasma and spectral interferences.     

CE-ICP-MS for studying interactions between metals and biomolecules

Metal-biomolecule interactions comprise an important research area in metallomics, and are significant for biology, medicine, pharmacy, nutrition, metabolism, and environmental science. Hybrid techniques are preferred for studying interactions between metals and biomolecules. Of all the separation techniques, capillary electrophoresis (CE) exhibits high resolution, minimal sample and reagent consumption, and rapid and efficient separations with minor disturbance of the existing equilibrium between the metal species and their biomolecular complexes. Inductively coupled plasma mass spectrometry (ICP-MS) presents high sensitivity to most of elements and offers multi-element detection.This article provides an overview of CE-ICP-MS for the study of metal-biomolecule interactions. We discuss applications of CE-ICP-MS to the study of interactions between metals or metalloids and natural ligands, such as humic substances or fulvic acids, and the interchange of metal complexes with metal species in metalloproteins.     

Investigation of the analytical performance of gliding spark source mass spectrometry (GSSMS) for the trace analysis of nonconducting materials

A radiofrequency (rf) spark discharge in vacuum developing across the surface of dielectrics – a so-called gliding spark – has been applied to the direct mass spectrometric trace analysis of nonconducting materials. The special configuration of the electrodes strengthened the electric field over the surface of a nonconducting sample and created optimum conditions for the sputtering and ionization of the sample material. Mass spectrometric investigations of the charge composition of atomic ion and molecular ion formation in radiofrequency gliding spark plasma showed a significant difference to that of the original rf spark discharge between two conducting electrodes. The analytical figures of merit (reproducibility, relative sensitivity factors and detection limits of chemical elements) of gliding spark source mass spectrometry have been studied by using the glass standard reference materials NIST SRM 610 and 611 for the determination of trace elements in glass matrix.     

Trends in sorption preconcentration combined with noble metal determination.

Nowadays much attention is being paid to the determination of trace amounts of noble metals in geological, industrial, biological and environmental samples. The most promising techniques, such as inductively coupled plasma atomic emission spectrometry (ICP-AES), inductively coupled plasma mass spectrometry (ICP-MS) and electrothermal atomic absorption spectrometry (ETAAS) are characterized by high sensitivity. However, the accurate determination of trace noble metals has been limited by numerous interferences generated from the presence of matrix elements. To decrease, or eliminate, these interferences, the sorption preconcentration of noble metals is often used prior to their instrumental detection. A great number of hyphenated methods of noble metal determination using sorption preconcentration have been developed. This review describes the basic types of available sorbents, preconcentration procedures and preparations of the sorbent to the subsequent determination of noble metals. The specific features of instrumental techniques and examples of ETAAS, FAAS, ICP-AES, ICP-MS determinations after the sorption preconcentration of noble metals are considered. The references cited here were selected mostly from the period 1996 - 2006.     

High performance analytical characterization of refractory metals

It is first shown what effects trace impurities generally exert on metal properties and why trace analysis is essential to modern applications of refractory metals in today's high technology. The effect of trace impurities in metals on complex systems like microelectronic components is also discussed.It is then shown, what principal analytical requirements are mandatory for trace characterization of refractory metals at levels of rising purity (4 N to 6 N). A survey of analytical methods for trace and ultratrace characterization of refractory metals is given including the following methods: flame and graphite furnace atomic absorption spectrometry, ICP and DCP-atomic emission spectrometry, X-ray fluorescence spectrometry, activation analysis, mass spectrometric methods, especially SIMS and GDMS.     

微量金属元素的毛细管电泳分析方法及应用

综述了毛细管电泳分析微量金属元素的基本原理、分离模式(CZE、MKEC、非水电泳、芯片分离等)、检测方法(紫外、荧光、化学发光、安培、电导、质谱联用技术)等的进展和该技术在环境、生物医学领域的研究与应用。引用文献94篇。     

Chemical separation and inductively coupled plasma–atomic emission spectrometric determination of seventeen trace metals in thorium oxide matrix using a novel extractant – Cyanex-923

Comprehensive studies have been carried out on the extraction behaviour of thorium matrix vis-a-vis 17 trace metallic elements using a novel extractant viz. Cyanex-923. The near total extraction of thorium and quantitative separation of these metals has been established using inductively coupled argon plasma–atomic emission spectrometry (ICP–AES). The recovery of few representative elements has been confirmed by radio-active tracer studies. The studies carried out here have enabled determination of μg/l amounts of all analyte elements with a precision of better than 1% RSD with prior chemical separation from as low as 1 g thorium sample in just five chemical extractions.     

一种改进的同步辐射X射线荧光原位分析人肝细胞胞质溶胶内金属蛋白分布的方法

对同步辐射X射线荧光分析技术(SBXRF)原位测定蛋白质经十二烷基硫酸铀．聚丙烯酰胺凝胶电泳(SDS-PAGE)分离后条带中微量元素的方法做了重要改进。通过增加一个于胶步骤，使得本底材料对光源的康普顿散射明显减弱，本底信号降低到约为湿胶的10％，从而可以用来测定蛋白条带内比锌轻的微量元素的相对含量。所提出的方法有望成为一种具有广泛应用前景的生物样品中微量元素的种态分析方法。对人肝细胞胞质溶胶内金属蛋白分布的研究结果表明：在15-95kDa的范围内有6个含锌蛋白，相对分子量分别为17．5、20．5、27、35、55及63kDa，其中63kDa蛋白为主要的含锌蛋白；此外至少还有4种分子量分别为20、23、43和83．5kDa的含铁蛋白分布于这个分子量范围内。     

Trace characterization of high-purity molybdenum and tungsten by electrothermal atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry, inductively coupled plasma mass spectrometry and total reflection X-ray fluorescence spectrometry involving analyte—matrix separation

A technique for the separation of 42 trace elements from up to 5 g of molybdenum and tungsten matrices was developed by means of the radiotracer technique. It is based on adsorption of the analyses on the cation exchanger Dowex 50 W x 9 from a 4% H₂O₂/0.01 mol 1⁻¹ HNO₃ solution followed by their elution with 15 ml of 4 mol I⁻¹ HNO₃ in the opposite flow direction. Both matrices were removed with a separation factor > 10⁴. The separation technique was applied to the analysis of these materials by electrothermal atomic absorption spectrometry, inductively coupled plasma atomic emission spectrometry, inductively coupled plasma mass spectrometry and total reflection X-ray fluorescence spetrometry. For all the determination methods used, the limits of detection are given and compared with those of other methods. With inductively coupled plasma mass spectrometry, for 22 of the 30 assayed elements, limits of detection at the sub-ng g⁻¹ level were achieved. The results are compared with those obtained by radiochemical neutron activation analysis in this work and by glow discharge mass spectrometry, secondary ion mass spectrometry, isotope dilution mass spectrometry and by solution spectrometric methods in other laboratories.     

Analysis for metallothioneins using coupled techniques

Analytical chemistry of metallothioneins based on the coupling of a high resolution separation technique with an element or species selective detection technique is discussed. The role of size-exclusion chromatography (SEC) with on-line atomic spectrometric detection for the quantification of metallothionein fraction in cell cytosols is evaluated. Particular attention is given to the conditions for the separation of metallated metallothionein isoforms (MT-1, MT-2, MT-3) and sub-isoforms within these classes by anion-exchange and reversed-phase HPLC. Techniques for interfacing chromatography with atomic absorption spectrometry (AAS), inductively coupled plasma atomic emission spectrometry (ICP AES) and ICP mass spectrometry (MS) are assessed. The potential of electrospray (tandem) mass spectrometry for the characterization of metallothionein isoforms with respect to molecular mass and aminoacid sequence is highlighted. Perspectives for capillary zone electrophoresis (CZE), microbore and capillary HPLC with ICP MS and electrospray MS(/MS) detection for the probing of metallothioneins are discussed. Applications of hyphenated techniques to the analysis of real-world samples are reviewed.     

Recent advances in on-line coupling of capillary electrophoresis to atomic absorption and fluorescence spectrometry for speciation analysis and studies of metal-biomolecule interactions

Speciation information is vital for the understanding of the toxicity, mobility and bioavailability of elements in environmental or biological samples. Hyphenating high resolving power of separation techniques and element-selective detectors provides powerful tools for studying speciation of trace elements in environmental and biological systems. During the last five years several novel hybrid techniques based on capillary electrophoresis (CE) and atomic spectrometry have been developed for speciation analysis and metal-biomolecule interaction study in our laboratory. These techniques include CE on-line coupled with atomic fluorescence spectrometry (AFS), chip-CE on-line coupled with AFS, CE on-line coupled with flame heated quartz furnace atomic absorption spectrometry (FHF-AAS), and CE on-line coupled with electrothermal atomic absorption spectrometry (ETAAS). The necessity for the development of these techniques, their interface design, and applications in speciation analysis and metal-biomolecule interaction study are reviewed. The advantages and limitations of the developed hybrid techniques are critically discussed, and further development is also prospected.     

Direct determination of trace elements in niobium,tantalum and their oxides by inductively coupled plasma atomic emission spectrometry after microwave dissolution

Analytical schemes for the determination of trace elements in high-purity niobium, tantalum and their oxides are proposed. The schemes are based on microwave dissolution of the metals and oxides followed by inductively coupled plasma atomic emission spectrometry (ICP-AES) determination of impurities in the solutions. The possibilities of interelement and off-peak background corrections in ICP-AES analysis are discussed. The accuracy of the results obtained is confirmed by the determination of trace elements after a matrix sorption separation procedure. For a number of elements, a comparison of the results obtained by ICP-AES without and with the matrix separation procedure and by electrothermal atomic absorption spectrometry (ETAAS) shows good agreement. The limits of detection for direct ICP-AES determination are in the range 0.4*1.0 μg g⁻¹ for Ba, Ca, Fe, Mg, Mn, Y and La; between 2.0 and 10.0 μ g⁻¹ for B, Cd, Co, Cr, Cu, Hf, Mo, Na, Nb, Ni, Pb, Sr, Ti, Zr and Ta; and for K, Sb and W a detection limit of 20 μ g⁻¹ is achieved. The schemes proposed are intended for rapid routine analysis.