The state-of-the-art of species-selective analysis for trace
metals and metalloids in biological materials by chromatographic and
electrophoretic separation techniques with element selective detection is
critically reviewed. The species of interest include organoarsenic,
organoselenium and metal complexes with bioligands such as phytochelatins,
metallothioneins, proteins and polysaccharides. The separation mechanisms
discussed include size-exclusion, anion- and cation-exchange and
reversed-phase HPLC and flatbed and capillary zone electrophoresis.
Advantages and limitations of various element selective (e.g.,
AAS, ICP-AES and ICP-MS) and molecule specific (electrospray MS/MS)
detection techniques used on- and off-line are discussed. The applications
of coupled techniques to the analysis of biological materials are
comprehensively reviewed in tabular form. Attention is paid to the sample
preparation and sources of error in bioinorganic speciation analysis.

Bio-inorganic speciation analysis by hyphenated techniques.

The emerging role of ICP-MS in proteomic analysis

Inductively coupled plasma mass spectrometric detection for chromatography and capillary electrophoresis

Analysis for metallothioneins using coupled techniques

A new method combining chemical modification and affinity purification is described for the characterization of serine and threonine phosphopeptides in proteins. The method is based on the conversion of phosphoserine and phosphothreonine residues to S-(2-mercaptoethyl)cysteinyl or β-methyl-S-(2-mercaptoethyl)cysteinyl residues by β-elimination/1,2-ethanedithiol addition, followed by reversible biotinylation of the modified proteins. After trypsin digestion, the biotinylated peptides were affinity-isolated and enriched, and subsequently subjected to structural characterization by liquid chromatography/tandem mass spectrometry (LC/MS/MS). Database searching allowed for automated identification of modified residues that were originally phosphorylated. The applicability of the method is demonstrated by the identification of all known phosphorylation sites in a mixture of α-casein, β-casein, and ovalbumin. The technique has potential for adaptations to proteome-wide analysis of protein phosphorylation. Copyright © 2001 John Wiley & Sons, Ltd.

Selective analysis of phosphopeptides within a protein mixture by chemical modification, reversible biotinylation and mass spectrometry.

A metal-binding metallothionein-like protein in a cyanobacterium, Anacystis nidulans R-2, has been spectated by high- performance gel permeation liquid chromatography combined with inductively coupled plasma mass spectrometry (HPLC-ICP/MS). Cadmium-stressed culturing induced a characteristic water-soluble protein in the cyanobacterium. The protein had an apparent molecular weight of ca. 10000, and contained predominantly cadmium as well as smaller amounts of copper and zinc. The cysteine residues in the protein were quantitated on 202Hg chromatograms after modifying the sulfhydryl groups with p-chloromercuribenzoate. The present findings demonstrate that the HPLC- ICP/MS technique has high potential for the trace level speciation of heavy metal-binding proteins of environmental interest in living organisms.

Application of High-Performance Liquid Chromatography/Inductively Coupled Plasma Mass Spectrometry to the Speciation of Cadmium-Binding Metallothionein-like Protein in a Cyanobacterium.

Selenium is a toxic element which can cause environmental pollution. Its uptake and incorporation by Zn2+-induced metallothionein (MT) biosynthesis in a cyanobacterium were studied by means of high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometric (ICPMS) detection. When we cultured the cells in selenite-enriched media, as much as 60% of the total selenium in the water-soluble cell components was incorporated into MT, which exists as two major isoforms but occupies a very minor fraction of the overall proteins. Existence of two pathways has been shown for the first time for selenium incorporation into the cyanobacterial MT; one is the S-Se bonding which can be reductively broken by 2-mercaptoethanol, and the other is the formation of selenocysteine followed by its incorporation into the MT peptide chain. No essential difference was noted between selenite and selenate regarding the uptake or the incorporation of Se. The present HPLC/ICPMS system can elucidate biological processes of environmental chemical importance at the molecular level.

HPLC/ICP Mass Spectrometric Study of the Selenium Incorporation into Cyanobacterial Metallothionein Induced under Heavy-Metal Stress.

High-performance liquid chromatography (HPLC) interface to inductively coupled plasma mass spectrometry (ICPMS) has been applied for the first time to determine the sulfhydryl groups (SHs) in chicken ovalbumin (OVA) after their conversion into mercaptides with organic mercury compounds. The procedure is based on the HPLC separation of mercury mercaptide OVA followed by ICPMS detection of mercury. The rates of reaction of five mercaptide-forming reagents with SHs in OVA were in the order ethylmercuric chloride > mersalyl acid > p-chloromercuribenzoate > p-(chloromercuri)benzenesulfonic acid > fluorescein mercuric acetate. Apparently the reaction rate is slowed by an increase in the steric hindrance around the mercury atom. The detection limit for SHs was lower than 1 pmol of OVA.

Determination of sulfhydryl groups in ovalbumin by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection.

The effects of Cu2+, Zn2+and Cd2+ ions on the induction of metallothionein (MT) in a cyanobacterium Anacystis nidulans R-2 have been studied by high-performance liquid chromatography (HPLC) with inductively coupled plasma mass spectrometric (ICP/MS) detection. After growing the cells in media of systematically varied metal concentrations, the contents of each metal in induced MT were determined by HPLC-ICP/MS. By separate dosing of a metal ion, MT induction was clearly observed with Zn2+and Cd2+, but not with Cu2+ However, Cu2+induced MT in the presence of a high level (0.77μM) of Zn2+ A high level of Zn2+ also significantly promoted MT induction by Cd2+. These synergistic effects have revealed the predominant role of Zn2+in heavy-metal detoxification by the organism. The partition of Cu, Zn and Cd between the cytoplasm and the cell wall was studied for cells grown in a medium fairly enriched in the three heavy-metal ions.

Individual and Synergistic Effects of Heavy-Metal Ions on the Induction of Cyanobacterial Metallothionein Examined by High-Performance Liquid Chromatography/Inductively Coupled Plasma Mass Spectrometry

By combining reversed-phase high-performance liquid chromatography (RP-HPLC) with inductively coupled plasma mass spectrometry (ICPMS), the induction of two major metallothionein (MT) isoforms has been demonstrated for a cyanobacterium Anacystis nidulans R-2, cultured under a stress of Zn2+ or Cd2+ The two isoforms showed only a slight difference in the amino acid composition Incubation of two Zn-MT isoforms in a medium containing Cd2+ led to a metal exchange, reflecting the higher affinity of MT to Cd2+ than to Zn2+ During this process the relative abundance of the two isoforms remained practically unchanged The two isoforms were induced in a constant ratio by the Zn2+ dosage (1-20μM), while by the Cd2+ dosage (05-10μM) the ratio showed a change with the dose level These results demonstrate the potential of the present RP-HPLC/ICPMS system to elucidate the function of MT isoforms

Tadashi Watanabe

Papers

Application of High-Performance Liquid Chromatography/Inductively Coupled Plasma Mass Spectrometry to the Speciation of Cadmium-Binding Metallothionein-like Protein in a Cyanobacterium.

HPLC/ICP Mass Spectrometric Study of the Selenium Incorporation into Cyanobacterial Metallothionein Induced under Heavy-Metal Stress.

Determination of sulfhydryl groups in ovalbumin by high-performance liquid chromatography with inductively coupled plasma mass spectrometric detection.

Individual and Synergistic Effects of Heavy-Metal Ions on the Induction of Cyanobacterial Metallothionein Examined by High-Performance Liquid Chromatography/Inductively Coupled Plasma Mass Spectrometry

Characterization and Quantitation of Metallothionein Isoforms Induced in a Cyanobacterium Using Reversed-Phase HPLC/ICP Mass Spectrometry