Development of Mercury Analysis by NanoSIMS for the Localization of Mercury-Selenium Particles in Whale Liver.

Nanoscale secondary ion mass spectrometry (NanoSIMS) is a dynamic SIMS technique, which offers high spatial resolution allowing the mapping of chemical elements at the nanometer scale combined with high sensitivity. However, SIMS for mercury analysis is a challenging issue due to the low secondary ion yield and has never been done on NanoSIMS. The introduction of an rf plasma oxygen primary ion source on NanoSIMS enabled higher lateral resolution and higher sensitivity for electropositive elements such as most metals. In this paper, for the first time, mercury analysis by NanoSIMS was developed applying the new rf plasma O- ion source. All mercury isotopes could be detected as Hg+ secondary ions and the isotopic pattern corresponded to their natural isotopic abundances. Furthermore, Hg+ detection in HgSe nanocrystals has been investigated where polyatomic interferences from selenium clusters were identified and separated by high mass resolution (ΔM/M ≥ 3200). However, in the presence of selenium a strong matrix effect was observed, decreasing the Hg+ secondary ion yield. In addition, a detection of Se+ ions was possible, too. The newly developed method was successfully applied to nanoscale localization by chemical imaging of HgSe particles accumulated in the liver tissue of sperm whale (Physeter macrocephalus). This demonstrated the applicability of NanoSIMS not only for mercury detection in surface analysis but also for mercury mapping in biological samples.

Pyrenoidal sequestration of cadmium impairs carbon dioxide fixation in a microalga.

Mixotrophic microorganisms are able to use organic carbon as well as inorganic carbon sources and thus, play an essential role in the biogeochemical carbon cycle. In aquatic ecosystems, the alteration of carbon dioxide (CO2 ) fixation by toxic metals such as cadmium - classified as a priority pollutant - could contribute to the unbalance of the carbon cycle. In consequence, the investigation of cadmium impact on carbon assimilation in mixotrophic microorganisms is of high interest. We exposed the mixotrophic microalga Chlamydomonas reinhardtii to cadmium in a growth medium containing both CO2 and labelled 13 C-[1,2] acetate as carbon sources. We showed that the accumulation of cadmium in the pyrenoid, where it was predominantly bound to sulphur ligands, impaired CO2 fixation to the benefit of acetate assimilation. Transmission electron microscopy (TEM)/X-ray energy dispersive spectroscopy (X-EDS) and micro X-ray fluorescence (µXRF)/micro X-ray absorption near-edge structure (µXANES) at Cd LIII- edge indicated the localization and the speciation of cadmium in the cellular structure. In addition, nanoscale secondary ion mass spectrometry (NanoSIMS) analysis of the 13 C/12 C ratio in pyrenoid and starch granules revealed the origin of carbon sources. The fraction of carbon in starch originating from CO2 decreased from 73 to 39% during cadmium stress. For the first time, the complementary use of high-resolution elemental and isotopic imaging techniques allowed relating the impact of cadmium at the subcellular level with carbon assimilation in a mixotrophic microalga.

Subcellular Localization of Copper-Cellular Bioimaging with Focus on Neurological Disorders.

As an essential trace element, copper plays a pivotal role in physiological body functions. In fact, dysregulated copper homeostasis has been clearly linked to neurological disorders including Wilson and Alzheimer's disease. Such neurodegenerative diseases are associated with progressive loss of neurons and thus impaired brain functions. However, the underlying mechanisms are not fully understood. Characterization of the element species and their subcellular localization is of great importance to uncover cellular mechanisms. Recent research activities focus on the question of how copper contributes to the pathological findings. Cellular bioimaging of copper is an essential key to accomplish this objective. Besides information on the spatial distribution and chemical properties of copper, other essential trace elements can be localized in parallel. Highly sensitive and high spatial resolution techniques such as LA-ICP-MS, TEM-EDS, S-XRF and NanoSIMS are required for elemental mapping on subcellular level. This review summarizes state-of-the-art techniques in the field of bioimaging. Their strengths and limitations will be discussed with particular focus on potential applications for the elucidation of copper-related diseases. Based on such investigations, further information on cellular processes and mechanisms can be derived under physiological and pathological conditions. Bioimaging studies might enable the clarification of the role of copper in the context of neurodegenerative diseases and provide an important basis to develop therapeutic strategies for reduction or even prevention of copper-related disorders and their pathological consequences.

A New Radio Frequency Plasma Oxygen Primary Ion Source on Nano Secondary Ion Mass Spectrometry for Improved Lateral Resolution and Detection of Electropositive Elements at Single Cell Level.

An important application field of secondary ion mass spectrometry at the nanometer scale (NanoSIMS) is the detection of chemical elements and, in particular, metals at the subcellular level in biological samples. The detection of many trace metals requires an oxygen primary ion source to allow the generation of positive secondary ions with high yield in the NanoSIMS. The duoplasmatron oxygen source is commonly used in this ion microprobe but cannot achieve the same quality of images as the cesium primary ion source used to produce negative secondary ions (C(-), CN(-), S(-), P(-)) due to a larger primary ion beam size. In this paper, a new type of an oxygen ion source using a rf plasma is fitted and characterized on a NanoSIMS50L. The performances of this primary ion source in terms of current density and achievable lateral resolution have been characterized and compared to the conventional duoplasmatron and cesium sources. The new rf plasma oxygen source offered a net improvement in terms of primary beam current density compared to the commonly used duoplasmatron source, which resulted in higher ultimate lateral resolutions down to 37 nm and which provided a 5-45 times higher apparent sensitivity for electropositive elements. Other advantages include a better long-term stability and reduced maintenance. This new rf plasma oxygen primary ion source has been applied to the localization of essential macroelements and trace metals at basal levels in two biological models, cells of Chlamydomonas reinhardtii and Arabidopsis thaliana.

Assessment of trace element and mineral levels in students from Turkmenistan in comparison to Iran and Russia.

THE OBJECTIVE: Of the present study was to assess essential trace element and mineral levels in serum, hair, and urine of healthy first-year students from Turkmenistan (n = 73) in comparison to students from Iran (n = 78) or Russia (n = 95). MATERIALS AND METHODS: Examination of foreign students was performed within two days after arrival to Russia during medical examination prior admission to RUDN University. Serum, hair, and urine trace element and mineral levels were assessed with inductively coupled plasma-mass spectrometry (ICP-MS). RESULTS: The data demonstrate that the levels of trace elements and minerals in students from Turkmenistan share high similarity to elemental profiles of students from Iran. In comparison to students from Russia, subjects originating from Iran and Turkmenistan are characterized by lower serum cobalt (Co), chromium (Cr), copper (Cu), manganese (Mn), molybdenum (Mo), selenium (Se), vanadium (V), zinc (Zn) levels, higher urinary Cr, Cu, Fe, Mn, V, and Zn, lower urinary Co and hair Mo, Se, and Zn content. Concomitantly, students from Turkmenistan were characterized by lower urinary Cr and Cu, serum Cu and V levels, higher circulating Zn concentration, as well as the lower hair Cr, Cu, iodine (I) and magnesium (Mg) content in comparison to Iranian subjects. The discriminant analysis demonstrated that hair, serum, and urinary trace element and mineral levels contributed to complete discrimination between the groups of students from different countries. CONCLUSIONS: The high similarity of trace element and mineral status of students from Turkmenistan and Iran is expected to be mediated by similar geochemical conditions in the bordering countries.

Nano-high-performance liquid chromatography with online precleaning coupled to inductively coupled plasma mass spectrometry for the analysis of lanthanide-labeled peptides in tryptic protein digests.

Low background signals are an indispensable prerequisite for accurate quantification in bioanalytics. This poses a special challenge when using derivatized samples, where excess reagent concentrations are increasing the background signal. Precleaning steps often are time-consuming and usually lead to analyte losses. In this study, a set of labeled model peptides and a protein digest was analyzed using inductively coupled plasma mass spectrometry (ICPMS), coupled to nano ion pairing reversed-phase high-performance liquid chromatography (nano-IP-RP-HPLC). In addition, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) was used for peptide identification. Peptides were labeled with lanthanide metals using bifunctional DOTA-based (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) reagents. The resulting metal excess was removed online during nano-HPLC, by trapping the labeled peptides on a C18-precolumn and washing them prior to their elution to the analytical column. Different ion pairing reagents like TFA (trifluoroacetic acid) and HFBA (heptafluorobutyric acid) were used in the study to enhance interactions of the different peptide species with the C18 material of the precolumn. HFBA even allowed the detection of a highly hydrophilic peptide that was not retained using TFA. It was shown that for the mixture of labeled model peptides, even a short 3 min washing step already enhanced the removal of the excess reagents significantly, whereas peptide losses were observable starting with a 10 min washing time. A 6 min washing time was determined to be the best parameter for lowering the lanthanide metal background while maintaining maximum peptide recovery. Alternative precleaning setups using EDTA to enhance the removal of free metal or an offline approach using solid phase extraction did not show promising results. The application of the optimized method to labeled peptides in a lysozyme digest showed results comparable to those obtained with model peptides.

Monitoring of protease catalyzed reactions by quantitative MALDI MS using metal labeling.

Quantitative mass spectrometry is a powerful tool for the determination of enzyme activities as it does not require labeled substrates and simultaneously allows for the identification of reaction products. However, major restrictions are the limited number of samples which can be measured in parallel due to the need for isotope labeled internal standards. Here we describe the use of metal labeling of peptides for the setup of multiplexed enzyme activity assays. After proteolytic reaction, using the protease trypsin, remaining substrates and peptide products formed in the reaction were labeled with metal chelators complexing rare earth metal ions. Labeled peptides were quantified with high accuracy and over a wide dynamic range (at least 2 orders of magnitude) using MALDI MS in case of simple peptide mixtures or by LC-MALDI MS for complex substrate mixtures and used for the monitoring of time-dependent product formation and substrate consumption. Due to multiplexing capabilities and accuracy, the presented approach will be useful for the determination of enzyme activities with a wide range of biochemical and biotechnological applications.

Metal labeling for accurate multiplexed peptide quantification via matrix-assisted laser desorption/ionization mass spectrometry.

Two peptide quantification strategies, the isobaric tags for relative or absolute quantitation (iTRAQ) labeling methodology and a metal-chelate labeling approach, were compared using matrix-assisted laser desorption/ionization-TOF/TOF MS and MS/MS analysis. Amino- and cysteine-directed labeling using the rare earth metal chelator 1,4,7,10-tetraazacyclododecane-N,N',N″,N″'-tetraacetic acid (DOTA) were applied for relative quantification of single peptides and a six-protein mixture. For analyte ratios close to one, iTRAQ and amino-directed DOTA labeling delivered overall comparable results regarding accuracy and reproducibility. In contrast, the MS-based quantification via amino-directed lanthanide-DOTA tags was more accurate for analyte ratios ≥5 and offered an extended dynamic range of three orders of magnitude. Our results show that the amino-directed DOTA labeling is an alternative relative quantification tool offering advantages like flexible multiplexing possibilities and, in particular, large dynamic ranges, which should be useful in sophisticated, targeted issues, where the accurate determination of extremely different protein or peptide concentration becomes relevant.

First insight in element localisation in different body parts of the acanthocephalan Dentitruncus truttae using TEM and NanoSIMS.

Acanthocephalans, intestinal parasites of vertebrates, are characterised by orders of magnitude higher metal accumulation than free-living organisms, but the mechanism of such effective metal accumulation is still unknown. The aim of our study was to gain new insights into the high-resolution localization of elements in the bodies of acanthocephalans, thus taking an initial step towards elucidating metal uptake and accumulation in organisms under real environmental conditions. For the first time, nanoscale secondary ion mass spectrometry (NanoSIMS) was used for high-resolution mapping of 12 elements (C, Ca, Cu, Fe, N, Na, O, P, Pb, S, Se, and Tl) in three selected body parts (trunk spines, inner part of the proboscis receptacle and inner surface of the tegument) of Dentitruncus truttae, a parasite of brown trout (Salmo trutta) from the Krka River in Croatia. In addition, the same body parts were examined using transmission electron microscopy (TEM) and correlated with NanoSIMS images. Metal concentrations determined using HR ICP-MS confirmed higher accumulation in D. truttae than in the fish intestine. The chemical composition of the acanthocephalan body showed the highest density of C, Ca, N, Na, O, S, as important and constitutive elements in living cells in all studied structures, while Fe was predominant among trace elements. In general, higher element density was found in trunk spines and tegument, as body structures responsible for substance absorption in parasites. The results obtained with NanoSIMS and TEM-NanoSIMS correlative imaging represent pilot data for mapping of elements at nanoscale resolution in the ultrastructure of various body parts of acanthocephalans and generally provide a contribution for further application of this technique in all parasite species.

Characteristic features of toxic metal content in hair samples of foreign students at RUDN University from different geographic regions.

BACKGROUND: This study explores the regional variations in toxic metal accumulation among RUDN University students from various global regions. METHODS: This comparative analysis examined hair samples from students hailing from different regions, including Russia, Asia, the Middle East, Africa, and Latin America. The concentrations of Aluminium (Al), Arsenic (As), Cadmium (Cd), Mercury (Hg), Lead (Pb), and Tin (Sn) were measured in the hair samples. The data was then evaluated using regression models to assess the link between the region of residence and toxic metal content in the hair. RESULTS: The analysis indicated significant regional variations in the levels of toxic metals in the students' hair. The highest content of Al, Cd, and Pb was observed in students from Africa (13.542, 0.028, 0.794 µg/g) and Latin America (9.947, 0.025, 0.435 µg/g). Arsenic levels in students from all regions exceeded that of Russian students by over two-fold. No substantial group differences were found in the Sn content. The regression models suggested that residing in Asia, Africa, and Latin America was a predictor of high Hg levels in hair (0.130, 0.096, 0.227 µg/g). Living in Africa was significantly associated with higher Pb levels (0.794 µg/g), and living in Latin America was close to significantly associated with the Cd level in the hair (0.025 µg/g). CONCLUSION: This study confirmed an increased accumulation of toxic metals, especially Hg, Cd, and Pb, in students primarily from Latin America and Africa. The findings highlighted the importance of understanding the regional variations in toxic metal accumulation to address associated health risks and the potential impact on students' well-being and academic performance. These insights may guide the development of targeted interventions to reduce exposure to toxic metals in students from various regions around the world.

High spatial resolution imaging of subcellular macro and trace element distribution during phagocytosis.

The bioavailability of trace elements in the course of evolution had an essential influence on the emergence of life itself. This is reflected in the co-evolution between eukaryotes and prokaryotes. In this study, the influence and cellular distribution of bioelements during phagocytosis at the host-pathogen interface were investigated using high-resolution nanoscale secondary ion mass spectrometry (NanoSIMS) and quantitative inductively coupled plasma mass spectrometry. In the eukaryotic murine macrophages (RAW 264.7 cell line), the cellular Fe/Zn ratio was found to be balanced, whereas the dominance of iron in the prokaryotic cells of the pathogen Salmonella enterica Serovar Enteritidis was ∼90% compared to zinc. This confirms the evolutionary increased zinc requirement of the eukaryotic animal cell. Using NanoSIMS, the Cs+ primary ion source allowed high spatial resolution mapping of cell morphology down to the subcellular level. At a comparable resolution, several low-abundant trace elements could be mapped during phagocytosis with a RF plasma O- primary ion source. An enrichment of copper and nickel could be detected in the prokaryotic cells. Surprisingly, an accumulation of cobalt in the area of the nuclear envelope was observed, indicating an interesting but still unknown distribution of this trace element in murine macrophages.

Comprehensive Speciation Analysis of Residual Gadolinium in Deep Cerebellar Nuclei in Rats Repeatedly Administered With Gadoterate Meglumine or Gadodiamide.

PURPOSE: Several preclinical studies have reported the presence of gadolinium (Gd) in different chemical forms in the brain, depending on the class (macrocyclic versus linear) of Gd-based contrast agent (GBCA) administered. The aim of this study was to identify, with a special focus on insoluble species, the speciation of Gd retained in the deep cerebellar nuclei (DCN) of rats administered repeatedly with gadoterate or gadodiamide 4 months after the last injection. METHODS: Three groups (N = 6/group) of healthy female Sprague-Dawley rats (SPF/OFA rats; Charles River, L'Arbresle, France) received a cumulated dose of 50 mmol/kg (4 daily intravenous administrations of 2.5 mmol/kg, for 5 weeks, corresponding to 80-fold the usual clinical dose if adjusted for man) of gadoterate meglumine (macrocyclic) or gadodiamide (linear) or isotonic saline for the control group (4 daily intravenous administrations of 5 mL/kg, for 5 weeks). The animals were sacrificed 4 months after the last injection. Deep cerebellar nuclei were dissected and stored at -80°C before sample preparation. To provide enough tissue for sample preparation and further analysis using multiple techniques, DCN from each group of 6 rats were pooled. Gadolinium species were extracted in 2 consecutive steps with water and urea solution. The total Gd concentrations were determined by inductively coupled plasma mass spectrometry (ICP-MS). Soluble Gd species were analyzed by size-exclusion chromatography coupled to ICP-MS. The insoluble Gd species were analyzed by single-particle (SP) ICP-MS, nanoscale secondary ion mass spectroscopy (NanoSIMS), and scanning transmission electron microscopy with energy-dispersive X-ray spectroscopy (STEM-EDX) for elemental detection. RESULTS: The Gd concentrations in pooled DCN from animals treated with gadoterate or gadodiamide were 0.25 and 24.3 nmol/g, respectively. For gadoterate, the highest amount of Gd was found in the water-soluble fractions. It was present exclusively as low-molecular-weight compounds, most likely as the intact GBCA form. In the case of gadodiamide, the water-soluble fraction of DCN was composed of high-molecular-weight Gd species of approximately 440 kDa and contained only a tiny amount (less than 1%) of intact gadodiamide. Furthermore, the column recovery calculated for this fraction was incomplete, which suggested presence of labile complexes of dissociated Gd3+ with endogenous molecules. The highest amount of Gd was detected in the insoluble residue, which was demonstrated, by SP-ICP-MS, to be a particulate form of Gd. Two imaging techniques (NanoSIMS and STEM-EDX) allowed further characterization of these insoluble Gd species. Amorphous, spheroid structures of approximately 100-200 nm of sea urchin-like shape were detected. Furthermore, Gd was consistently colocalized with calcium, oxygen, and phosphorous, strongly suggesting the presence of structures composed of mixed Gd/Ca phosphates. No or occasional colocalization with iron and sulfur was observed. CONCLUSION: A dedicated analytical workflow produced original data on the speciation of Gd in DCN of rats repeatedly injected with GBCAs. The addition, in comparison with previous studies of Gd speciation in brain, of SP element detection and imaging techniques allowed a comprehensive speciation analysis approach. Whereas for gadoterate the main fraction of retained Gd was present as intact GBCA form in the soluble fractions, for linear gadodiamide, less than 10% of Gd could be solubilized and characterized using size-exclusion chromatography coupled to ICP-MS. The main Gd species detected in the soluble fractions were macromolecules of 440 kDa. One of them was speculated to be a Gd complex with iron-binding protein (ferritin). However, the major fraction of residual Gd was present as insoluble particulate species, very likely composed of mixed Gd/Ca phosphates. This comprehensive Gd speciation study provided important evidence for the dechelation of linear GBCAs and offered a deeper insight into the mechanisms of Gd deposition in the brain.

Physiological characterization of cadmium-exposed Chlamydomonas reinhardtii.

Chlamydomonas reinhardtii is a common model organism for investigation of metal stress. This green alga produces phytochelatins in the presence of metal ions. The influence of cadmium is of main interest, because it is a strong activator of phytochelatin synthase. Cell wall bound and intracellular cadmium content was determined after exposition to 70 µm CdCl(2), showing the main portion of the metal outside the cell. Nevertheless, imported cadmium was sufficient to cause significant changes in thiolpeptide metabolism and its transcriptional regulation. Modern analytical approaches enable new insights into phytochelatin (PC) distribution. A new rapid and precise UPLC-MS method allowed high-throughput PC quantification in algal samples after 1, 4, 24 and 48 h cadmium stress. Initially, canonic PCs were synthesized in C. reinhardtii during cadmium exposition, but afterwards CysPCs became the major thiolpeptides. Thus, after 48 h the concentration of the PC-isoforms CysPC(2-3) and CysGSH attained between 105 and 199 nmol g(-1) fresh weight (FW), whereas the PC(2-3) concentrations were only 15 nmol g(-1) FW. The relative quantification of γ-glutamyl transpeptidase (γ-GT) mRNA suggests the generation of CysPCs by glutamate cleavage from canonic PCs by γ-GT. Furthermore, a homology model of C. reinhardtii phytochelatin synthase was constructed to verify the use of crystal structures from Anabaena sp. phytochelatin synthase (PCS) for docking studies with canonical PCs and CysPCs. From the difference in energy scores, we hypothesize that CysPC may prevent the synthesis of canonical PCs by blocking the binding pocket. Finally, possible physiological reasons for the high abundance of CysPC compared with their canonic precursors are discussed.

Recent directions of electrospray mass spectrometry for elemental speciation analysis.

A brief survey is given of the last 2 years' literature on electrospray mass spectrometry (ESI-MS) for speciation analysis. As observed for many years, the main recent applications in this field concern arsenic and selenium species, especially in studies encompassing combined use of molecular and element mass spectrometry. A further application field is the stoichiometric characterization of metal complexes by ESI-MS, which in some studies was assisted by nuclear magnetic resonance spectroscopy. A few examples are presented to illustrate arsenic species involved in metabolic pathways, sulfur species in oils and bitumen, and aluminum complexes. On the basis of this review, we also give an outlook of expected future developments and trends in this research field.

Characterization of metal-labelled peptides by matrix-assisted laser desorption/ionization mass spectrometry and tandem mass spectrometry.

Metal labelling of peptides and proteins using high-affinity metal-chelating compounds has found widespread applications in the medical and bioanalytical fields. In the present study we investigated the analysis of peptides derivatized either with cysteine- or amino group-directed metal-bound DOTA (1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid) chelators in matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). The metal complexes of DOTA were shown to be stable under MALDI-MS conditions. The introduction of the metal label led in a number of cases to significantly increased signal-to-noise (S/N) values and thus improved sensitivity of the labelled peptides compared to their unlabelled counterparts, especially for multiply labelled peptides. The presence of the labels did alter the tandem mass spectrometric (MS/MS) behaviour, namely the formation of sequence specific a-, b- and y-ion series, in dependence of the position of the label within the peptide sequence. For cysteine-derivatized peptides several label-specific reporter ions and characteristic immonium ions could be identified. Amino-directed labelling led only to the formation of characteristic immonium ions in ε-amino groups of lysine, whereas N-terminal labelling in some cases led to the formation of a(1)- and b(1)-ions. The results clearly show that MALDI-MS is suitable for the analysis of metal-labelled peptides, which was also confirmed in liquid chromatography (LC)/MALDI-based identification of proteins in a model protein mixture labelled with Cys-reactive DOTA. Here, in comparison to a run with alkylated cysteines, more than 50% more cysteine-containing peptides were identified.

Rapid and simple UPLC-MS/MS method for precise phytochelatin quantification in alga extracts.

Quantitative phytochelatin (PC) analysis is, due to oxidation sensitivity of the PCs, matrix effects, and time consuming sample preparation, still a challenging analytical task. In this study, a rapid, simple, and sensitive method for precise determination of native PCs in crude extracts of the green alga Chlamydomonas reinhardtii was developed. Algae were exposed 48 h to 70 µM Cd. Coupling of ultra performance liquid chromatography and electrospray ionization tandem mass spectrometry with multi-reaction mode transitions for detection permitted the required short-time, high-resolution separation and detection specificity. Thus, under optimized chromatographic conditions, 10 thiol peptides were baseline-separated within 7 min. Relative detection limits in the nanomolar range in microliter sample volumes were achieved (corresponding to absolute detection limits at femtomole level). Next to glutathione (GSH), the most abundant cadmium-induced PCs in C. reinhardtii, namely CysGSH, PC(2), PC(3), CysPC(2), and CysPC(3), were quantified with high reproducibility at concentrations between 15 and 198 nmol g(-1) fresh weight. The biological variation of PC synthesis of nine independently grown alga cultures was determined to be on average 13.7%.

Inhibitory effect of metals on animal and plant glutathione transferases.

Glutathione transferases (GSTs) represent a widespread enzyme superfamily in eukaryotes and prokaryotes catalyzing different reactions with endogenous and xenobiotic substrates such as organic pollutants. The latter are often found together with metal contamination in the environment. Besides performing of essential functions, GSTs protect cells by conjugation of glutathione with various reactive electrophiles. The interference of toxic metals with this functionality of GSTs may have unpredictable toxicological consequences for the organisms. In this review results from the recent literature are summarized and discussed describing the ability of metals to inhibit intracellular detoxification processes in animals and plants.

Absolute peptide quantification by lutetium labeling and nanoHPLC-ICPMS with isotope dilution analysis.

The need of analytical methods for absolute quantitative protein analysis spurred research on new developments in recent years. In this work, a novel approach was developed for accurate absolute peptide quantification based on metal labeling with lutetium diethylenetriamine pentaacetic acid (Lu-DTPA) and nanoflow high-performance liquid chromatography-inductively coupled plasma isotope dilution mass spectrometry (nanoHPLC-ICP-IDMS). In a two-step procedure peptides were derivatized at amino groups with diethylenetriamine pentaacetic anhydride (DTPAA) followed by chelation of lutetium. Electrospray ionization mass spectrometry (ESI MS) of the reaction product demonstrated highly specific peptide labeling. Under optimized nanoHPLC conditions the labeled peptides were baseline-separated, and the excess labeling reagent did not interfere. A 176Lu-labeled spike was continuously added to the column effluent for quantification by ICP-IDMS. The recovery of a Lu-DTPA-labeled standard peptide was close to 100% indicating high labeling efficiency and accurate absolute quantification. The precision of the entire method was 4.9%. The detection limit for Lu-DTPA-tagged peptides was 179 amol demonstrating that lutetium-specific peptide quantification was by 4 orders of magnitude more sensitive than detection by natural sulfur atoms present in cysteine or methionine residues. Furthermore, the application to peptides in insulin tryptic digest allowed the identification of interfering reagents decreasing the labeling efficiency. An additional advantage of this novel approach is the analysis of peptides, which do not naturally feature ICPMS-detectable elements.

Analytical approach for characterization of cadmium-induced thiol peptides--a case study using Chlamydomonas reinhardtii.

Phytochelatins (PC) were described earlier to play a role in metal detoxification in Chlamydomonas reinhardtii but were not clearly identified. The focus of this case study was to identify PC synthesized by C. reinhardtii exposed to Cd. Only low intracellular concentrations of cadmium (85 nmol g(-1) fresh weight) were sufficient to cause significant changes in thiol peptide pools. Thus, results showed a progressive decline of the glutathione content, accompanied by an induction of phytochelatins. Not only canonic phytochelatins but for the first time also the iso-phytochelatins CysPC(n) and PC(2)Ala were identified in this unicellular green alga using electrospray ionization quadrupole time-of-flight tandem mass spectrometry. Additionally, CysPC(n)desGly, PC(n)desGly, CysPC(n)Glu, and PC(2)Glu were found throughout MS analysis. Also, low abundant PCs could be detected due to the high sample preconcentration combined with little sample amounts (0.3 microL min(-1)) necessary for electrospray. Identified PCs had a maximum number of 5 gamma-glutamyl cysteine (gamma-GluCys) units. Thiol peptides of higher molecular masses suggesting PC(n) with n > 5 could be identified as intermolecular oxidation products of smaller PCs. Thiols may easily be oxidized. Therefore, PCs were reduced prior to MS analysis. Dithiothreitol and tris(2-carboxyethyl) phosphine were compared concerning their reduction effort.

Pb nanospheres in ancient zircon yield model ages for zircon formation and Pb mobilization.

Nanospheres of lead (Pb) have recently been identified in zircon (ZrSiO4) with the potential to compromise the veracity of U-Pb age determinations. The key assumption that the determined age is robust against the effects of Pb mobility, as long as Pb is not lost from the zircon during subsequent geological events, is now in question. To determine the effect of nanosphere formation on age determination, and whether analysis of nanospheres can yield additional information about the timing of both zircon growth and nanosphere formation, zircons from the Napier Complex in Enderby Land, East Antarctica, were investigated by high-spatial resolution NanoSIMS (Secondary Ion Mass Spectrometry) mapping. Conventional SIMS analyses with >µm resolution potentially mixes Pb from multiple nanospheres with the zircon host, yielding variable average values and therefore unreliable ages. NanoSIMS analyses were obtained of 207Pb/206Pb in nanospheres a few nanometres in diameter that were resolved from 207Pb/206Pb measurements in the zircon host. We demonstrate that analysis for 207Pb/206Pb in multiple individual Pb nanospheres, along with separate analysis of 207Pb/206Pb in the zircon host, can not only accurately yield the age of zircon crystallization, but also the time of nanosphere formation resulting from Pb mobilization during metamorphism. Model ages for both events can be derived that are correlated due to the limited range of possible solutions that can be satisfied by the measured 207Pb/206Pb ratios of nanospheres and zircon host. For the Napier Complex zircons, this yields a model age of ca 3110 Ma for zircon formation and a late Archean model age of 2610 Ma for the metamorphism that produced the nanospheres. The Nanosphere Model Age (NMA) method constrains both the crystallization age and age of the metamorphism to ~±135 Ma, a significant improvement on errors derived from counting statistics.