Size Exclusion Chromatography-inductively Coupled Plasma-mass Spectrometry Research Articles

Insights into copper sensing and tolerance in Pneumocystis species

IntroductionPneumocystis species are pathogenic fungi known to cause pneumonia in immunocompromised mammals. They are obligate to their host, replicate extracellularly in lung alveoli and thrive in the copper-enriched environment of mammalian lungs. In this study, we investigated the proteome of Pneumocystis murina, a model organism that infects mice, in the context of its copper sensing and tolerance.Methods and resultsThe query for copper-associated annotations in FungiDB followed by a manual curation identified only 21 genes in P. murina, significantly fewer compared to other clinically relevant fungal pathogens or phylogenetically similar free-living fungi. We then employed instrumental analyses, including Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS), Immobilized Metal Affinity Chromatography (IMAC), and Liquid Chromatography–Tandem Mass Spectrometry (LC–MS/MS), to isolate and identify copper-binding proteins from freshly extracted organisms, revealing 29 distinct cuproproteins. The RNA sequencing (RNA-seq) analysis of P. murina exposed to various CuSO4 concentrations at three temporal intervals (0.5, 2, and 5 h) indicated that significant gene expression changes occurred only under the highest CuSO4 concentration probed (100 μM) and the longest exposure duration (5 h). This stimulus led to the upregulation of 43 genes and downregulation of 27 genes compared to untreated controls. Quantitative PCR (qPCR) confirmed the expression of four out of eight selected upregulated genes, including three assumed transcription factors (PNEG_01236, PNEG_01675, and PNEG_01730) and a putative copper transporter (PNEG_02609). Notably, the three applied methodologies — homology-based annotation, SEC-ICP-MS/IMAC/LC–MS/MS, and RNA-seq — yielded largely distinct findings, with only four genes (PNEG_02587, PNEG_03319, PNEG_02584, and PNEG_02989) identified by both instrumental methods.DiscussionThe insights contribute to the broader knowledge of Pneumocystis copper homeostasis and provide novel facets of host-pathogen interactions for extracellular pathogens. We suggest that future studies of Pneumocystis pathogenicity and copper stress survival should consider the entire spectrum of identified genes.

Speciation Analysis of Gadolinium in the Water-Insoluble Rat Brain Fraction After Administration of Gadolinium-Based Contrast Agents.

To date, the analysis of gadolinium (Gd) speciation in the brain of animals administered with macrocyclic and linear Gd-based contrast agents (GBCAs) has been limited to Gd soluble in mild buffers. Under such conditions, less than 30% of the brain tissue was solubilized and the extraction recoveries of GBCAs into the aqueous phase were poor, especially in the case of the linear GBCAs. The aim of this study was to find the conditions to solubilize the brain tissue (quasi-)completely while preserving the Gd species present. The subsequent analysis using size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) was intended to shed the light on the speciation of the additionally recovered Gd. Four groups of healthy female Sprague Dawley rats (SPF/OFA rats; Charles River, L'Arbresle, France) received randomly 5 intravenous injections (1 injection per week during 5 consecutive weeks) of either gadoterate meglumine, gadobenate dimeglumine, gadodiamide (cumulated dose of 12 mmol/kg), or no injection (control group). The animals were sacrifice 1 week (W1) after the last injection. Brain tissues were solubilized with urea solution, whereas tissues extracted with water served as controls. Total Gd concentrations were determined in the original brain tissue and its soluble and insoluble fractions by inductively coupled plasma-mass spectrometry (ICP-MS) to calculate the Gd accumulation and extraction efficiency. Size exclusion chromatography coupled to ICP-MS was used to monitor the speciation of Gd in the soluble fractions. The stability of GBCAs in the optimum conditions was monitored by spiking the brain samples from the untreated animals. The column recoveries were precisely determined in the purpose of the discrimination of weakly and strongly bound Gd complexes. The identity of the eluted species was explored by the evaluation of the molecular size and retention time matching with Gd chelates and ferritin standard. The speciation analyses were carried out for 2 different brain structures, cortex and cerebellum. The combination of water and urea extractions (sequential extraction) managed to solubilize efficiently the brain tissue (97% ± 1%) while preserving the stability of the initially injected form of GBCA. For macrocyclic gadoterate, 97% ± 1% and 102% ± 3% of Gd initially present in the cortex and cerebellum were extracted to the soluble fraction. For gadobenate, similar amounts of Gd (49% ± 1% and 46% ± 4%) were recovered from cortex and cerebellum. For gadodiamide, 48% ± 2% of Gd was extracted from cortex and 34% ± 1% from cerebellum. These extraction efficiencies were higher than reported elsewhere. The SEC-ICP-MS and the column recovery determination proved that Gd present at low nmol/g levels in brain tissue was exclusively in the intact GBCA form in all the fractions of brain from the animals treated with gadoterate. For the linear GBCAs (gadobenate and gadodiamide), 3 Gd species of different hydrodynamic volumes were detected in the urea-soluble fraction: (1) larger than 660 kDa, (2) approximately 440 kDa, and (3) intact GBCAs. The species of 440 kDa corresponded, on the basis of the elution volume, to a Gd3+ complex with ferritin. Gd3+ was also demonstrated by SEC-ICP-MS to react with the ferritin standard in 100 mM ammonium acetate (pH 7.4). In contrast to macrocyclic gadoterate, for linear GBCAs, the column recovery was largely incomplete, suggesting the presence of free, hydrolyzed, or weakly bound Gd3+ with endogenous ligands. The sequential extraction of rat brain tissue with water and urea solution resulted in quasi-complete solubilization of the tissue and a considerable increase in the recoveries of Gd species in comparison with previous reports. The macrocyclic gadoterate was demonstrated to remain intact in the brain 1 week after administration to rats. The linear GBCAs gadobenate and gadodiamide underwent ligand exchange reactions resulting in the presence of a series of Gd3+ complexes of different strength with endogenous ligands. Ferritin was identified as one of the macromolecules reacting with Gd3+. For the linear GBCAs, 3% of the insoluble brain tissue was found to contain more than 50% of Gd in unidentified form(s).

Development of a method for the detection of zinc in Brassica oleracea using solid phase extraction and size-exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS)

The aim of this work is the development of a suitable method to extract and detect zinc-bound compounds from cabbage, broccoli and kale (family Brassicaceae, species oleracea) using solid phase extraction (SPE) and size-exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Tris [2-Amino-2-(hydroxymethyl)-1,3-propanediol]/hydrochloric acid (Tris/HCl) or ammonium nitrate were used as extractants added to the freeze-dried samples, which were then sonicated and centrifuged. An enzymatic mixture was added to the extracts and then incubated for 5- and 18 h prior to analysis by SEC-ICP-MS. Results showed a good coefficient of variation (CV) of the elution time (0.06-0.9%), concentration (4.7–16.9%) and molecular size (0.4–5.4%). The limit of detection (LOD) and the limit of quantitation (LOQ) were 0.9 μg L−1 and 2.8 μg L−1, respectively. The proposed method is reliable and robust and can be applied to samples with difficult matrices like vegetables and soil.•Good precision, stability and reproducibility.•Easy to execute and suitable for analysis of vegetables and other samples with complex matrices, eg. soil.•This method contributed to good maintenance of the instrument and to minimal cleaning time.

Quantification of ferritin-bound iron in murine samples for Alzheimer’s disease studies using species-specific isotope dilution mass spectrometry

We have investigated species-specific isotope dilution mass spectrometry (IDMS) for the quantification of ferritin-bound iron in murine serum and brain. Therefore, fresh samples were analyzed using size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS). Isotopically labeled (57Fe)ferritin was used as calibrant for the quantification of ferritin-bound iron in murine samples. Assessment of the iron load of serum ferritin was impaired by concomitant iron-containing proteins of similar size and shape, which could not be separated by SEC nor centrifugal ultra-filtration. In contrast, ferritin was the main iron-containing protein in cytosolic extracts of murine brain, which showed a total ferritin-bound iron content of (1.05 ± 0.12) µg g−1 (n= 10; U, k= 2). The relative expanded uncertainty achieved for a mass fraction of ca. 1 µg g−1 ferritin-bound iron was 11% (Urel, k = 2). The relative expanded uncertainty of the iron mass fraction of the (57Fe)ferritin spike was 5.7% and represented the major contributing factor to the overall uncertainty. Statistical tests suggested no significant difference in ferritin-bound iron content between mouse brain hemispheres. The presented analytical tool provides low limits of quantification (2.2 ng g−1) and uncertainties (11%, Urel, k = 2), thus enables the quantification of ferritin-bound iron in murine brain extracts with high sensitivity and accuracy. Furthermore, this analytical workflow assures comparability of measurement results across research laboratories. This provides the basis for investigation of the iron loading of ferritin in brain tissue of healthy and Alzheimer’s disease mouse models, which may help answering the question if iron regulation is impaired in Alzheimer’s disease.

Zinc Transporter-3 Knockout Mice Demonstrate Age-Dependent Alterations in the Metalloproteome

Metals are critical cellular elements that are involved in a variety of cellular processes, with recent literature demonstrating that zinc, and the synaptic zinc transporter (ZnT3), are specifically involved in learning and memory and may also be key players in age-related neurodegenerative disorders such as Alzheimer’s disease. Whilst the cellular content and location of metals is critical, recent data has demonstrated that the metalation state of proteins is a determinant of protein function and potential toxicity. As we have previously reported that ZnT3 knockout (KO) mice have deficits in total zinc levels at both 3 and 6 months of age, we were interested in whether there might be changes in the metalloproteomic profile in these animals. To do this, we utilised size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) and examined hippocampal homogenates from ZnT3 KO and age-matched wild-type mice at 3, 6 and 18 months of age. Our data suggest that there are alterations in specific metal binding proteins, for zinc, copper and iron all being modulated in the ZnT3 KO mice compared to wild-type (WT). These data suggest that ZnT3 KO mice may have impairments in the levels or localisation of multiple transition metals, and that copper- and iron-dependent cellular pathways may also be impacted in these mice.

Investigation of total zinc contents and zinc-protein profile in medicinal plants traditionally used for diabetes treatment.

This study aims at the assessment of total zinc contents, water zinc extract contents and zinc-protein profile in medicinal plants traditionally used for diabetes treatment. While zinc-protein profile was screened in plant samples using the online coupling of size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS), total zinc contents and zinc water extract contents were determined using inductively coupled plasma-optical emission spectrometry (ICP-OES). The analysis of two certified reference materials with similar matrix for total zinc content revealed recovery values of 97.1% and 100.5% while the average of recovery of the summed Zn concentrations from protein fractions compared to total Zn was 103.0 ± 4.8%. Based on the FAO/WHO classification, Sesamum indicum, Nigella sativa, Trigonella Foenum-graecum and Pennisetum glaucum are classified as highest Zn-content. For protein profile, zinc was quantified in 330-430 and 50-60kDa fractions of all examined plants while no contents were quantified in the inorganic fraction 0.05-0.4kDa of all plant species. Also, only three plant species recorded Zn contents in the phytate fraction (0.9-1.5kDa fraction). The fruits of the Momordica Charantia and the Citrullus colocynthis were with the highest extractable zinc concentration; 13.55 ± 0.45 and 10.08 ± 0.63mg/kg, respectively. The highest Zinc capturing capacity was reported for the 50-60 and 70-87 KDa protein fractions.

Temperature‐dependent irreversible conformational change of recombinant ADAMTS13 upon metal ion chelation

BackgroundThe catalytic domain of ADAMTS13 possesses one Zn2+ and up to three putative Ca2+ binding sites and can be inactivated by chelating agents. Although replenishment with an appropriate metallic cation is thought to restore the enzyme's proteolytic activity fully, ADAMTS13 stability in a metal ion‐depleting environment has not been explored. ObjectivesTo address the stability of ADAMTS13 in citrated human plasma. MethodsADAMTS13 activity was measured using the FRETS‐VWF73 fluorogenic assay. The molar ratio of metals bound to ADAMTS13 was determined by size exclusion chromatography inductively coupled plasma mass spectrometry (SEC‐ICP‐MS). Higher‐order structural changes were analyzed using Fourier‐transformed infrared spectroscopy and dynamic light scattering. ResultsADAMTS13 was stable at room temperature for up to 24 hours irrespective of the presence of citrate (0.38%). However, at 37°C, citrate caused a time‐dependent activity decrease. No ADAMTS13 activity decrease was seen in heparinized plasma, but the addition of citrate again caused ADAMTS13 instability at 37°C. Scavenging of citrate by the addition of Ca2+ or Zn2+ prior to but not postincubation prevented the activity decrease of the enzyme. The SEC‐ICP‐MS analyses showed that ADAMTS13 only bound Zn2+ and that its reduced activity correlated with a gradual loss of bound Zn2+. Concomitant higher‐order structural analyses demonstrated structural changes in ADAMTS13 that are typical of less‐ordered protein structures. ConclusionsZn2+ is required to stabilize ADAMTS13 structure at physiologic temperature, thereby preventing irreversible loss of enzyme activity. This finding is particularly important to consider when using citrated human plasma as a source of ADAMTS13 in clinical settings.

SEC-ICP-MS and on-line isotope dilution analysis for characterisation and quantification of immunochemical assays.

This study presents a novel size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method for the characterisation and quantification of immunoassays with lanthanide-labelled antibodies. SEC-ICP-MS in combination with a double isotope dilution approach enabled facile validation of the antibodies' integrity, the determination of the batch to batch labelling efficiency, monitoring of each labelling step, and quantification of the immunocomplexes after incubation with the target protein. The addition of oxygen into the dynamic reaction cell improved the detection of sulphur as a marker for the antibodies and target protein via mass-shifting (LOD = 3.7ng/mL), whilst maintaining sufficient sensitivity for the analysis of the lanthanides. Ultra-high performance liquid chromatography (UHPLC) SEC ensured a rapid chromatographic method with separation times under 7min of the labelled and unlabelled antibodies, the immunocomplexes, and the unconjugated polymer used to lanthanide-label the antibodies. SEC calibration estimated the molecular weights of all peaks and provided valuable insights in immunochemical reactions and the stoichiometry of the reactants and products. A novel on-line isotope dilution analysis (IDA) enabled absolute quantification of sulphur and lanthanide signals and the protein of interest. The chromatographic separation of immunocomplexes and labelled antibodies allowed the simultaneous determination of the antibody/metal stoichiometry and target protein concentration from a single mass flow chromatogram. An immunoglobulin protein was quantified after incubation with an 153Eu-labelled primary polyclonal antibody. The procedure was validated with direct labelling of the target protein with 156Gd for parallel, simultaneous quantification. The concentration determined via direct labelling of the protein deviated 1.9% from the immunochemical approach employing 153Eu-labelled polyclonal antibodies. Graphical abstract.

Optimization of Microwave-Ultrasound-assisted Enzymatic Hydrolysis extraction of iodine amino acids in laminaria by high performance liquid chromatography with a photodiode array detector

A rapid and efficient method for extracting Mnoiodo-tyrosine (MIT) and Diiodo-tyrosine (DIT) from laminaria was successfully developed. The laminaria samples were first pretreated with microwaves, before the MIT and DIT were extracted with an ultrasonic water bath. A response surface experiment was used to optimize the extraction conditions, and ultimately the best conditions for extraction were successfully identified, which were a microwave power of 250 W, a microwave time of 4.7 min, an addition of 0.31 g of pancreatin, and 4.22 h in the ultrasonic water bathe. A simple and low-cost High-Performance Liquid Chromatography High-Performance Liquid Chromatography Photodiode Array (HPLC-PDA) detection method for MIT and DIT was established. The detection limits ranged from 50.6 to 320.6 ng/g. Scanning Electron Microscopy (SEM) was used to observe the degree of damage to the laminaria cells from the different extraction methods. The molecular weight distribution of the organic iodine of the original laminaria and the laminaria after enzymatic hydrolysis was studied by Size-Exclusion Chromatography Inductively Coupled Plasma Mass Spectrometry (SEC-ICP-MS).

Selenium Levels in Serum, Red Blood Cells, and Cerebrospinal Fluid of Alzheimer's Disease Patients: A Report from the Australian Imaging, Biomarker & Lifestyle Flagship Study of Ageing (AIBL).

Selenium (Se) protects cells against oxidative stress damage through a range of bioactive selenoproteins. Increased oxidative stress is a prominent feature of Alzheimer's disease (AD), and previous studies have shown that Se deficiency is associated with age-related cognitive decline. In this study, we assessed Se status in different biofluids from a subgroup of participants in the Australian Imaging, Biomarkers and Lifestyle Flagship Study of Ageing. As Se in humans can either be an active component of selenoproteins or inactive via non-specific incorporation into other proteins, we used both size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and tandem mass spectrometry to characterize selenoproteins in serum. We observed no differences in total Se concentration in serum or cerebrospinal fluid of AD subjects compared to mildly cognitively impairment patients and healthy controls. However, Se levels in erythrocytes were decreased in AD compared to controls. SEC-ICP-MS analysis revealed a dominant Se-containing fraction. This fraction was subjected to standard protein purification and a bottom-up proteomics approach to confirm that the abundant Se in the fraction was due, in part, to selenoprotein P. The lack of change in the Se level is at odds with our previous observations in a Brazilian population deficient in Se, and we attribute this to the Australian cohort being Se-replete.

Cadmium Distribution and Characteristics of Cadmium-binding Proteins in Rice (<i>Oryza sativa L.</i>) Kernel

Studies were performed to investigate the Cadmium (Cd) distribution and Cd-binding proteins in rice kernel. Rice seeds [Oryza sativa L. (Akitakaomachi)] were cultured under Cd stress and after harvesting, the Cd distribution was determined using laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS). The Cd concentrations and amino acid compositions in different storage protein fractions were analyzed using ICP-MS and an amino acid analyzer, respectively. The molecular weights of the Cd-binding proteins were determined by size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS). The Cd concentrations in rice kernel gradually decreased from the outer bran layers to the endosperm and Cd was particularly concentrated in the embryo, the outer pericarp and the aleurone layer. The Cd concentrations in different proteins fractions decreased in the following sequence: globulin > albumin > glutelin > prolamin. The presence of cysteine and methionine were closely related to the Cd concentration. The molecular weights of three prominent Cd-binding protein fractions were > 2000 k Da, 570 k Da and 131.9 k Da, respectively.

Trace element-protein interactions in endolymph from the inner ear of fish: implications for environmental reconstructions using fish otolith chemistry.

Otoliths, the biomineralised hearing "ear stones" from the inner ear of fish, grow throughout the lifespan of an individual, with deposition of alternating calciferous and proteinaceous bands occurring daily. Trace element : calcium ratios within daily increments measured by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) are often used in fisheries science to reconstruct environmental histories. There is, however, considerable uncertainty as to which elements are interacting with either the proteinaceous or calciferous zones of the otolith, and thus their utility as indicators of environmental change. To answer this, we used size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) of endolymph, the otolith growth medium, to determine the binding interactions for a range of elements. In addition, we used solution ICP-MS to quantify element concentrations in paired otolith and endolymph samples and determined relative enrichment factors for each. We found 12 elements that are present only in the proteinaceous fraction, 6 that are present only in the salt fraction, and 4 that are present in both. These findings have important implications for the reconstruction of environmental histories based on changes in otolith elemental composition: (1) elements occurring only in the salt fraction are most likely to reflect changes in the physico-chemical environment experienced during life; (2) elements occurring only in the proteinaceous fraction are more likely to reflect physiological rather than environmental events; and (3) elements occurring in both the salt and proteinaceous fractions are likely to be informative about both endogenous and exogenous processes, potentially reducing their utility in environmental reconstructions.

Changes in Brain Metallome/Metabolome Pattern due to a Single i.v. Injection of Manganese in Rats.

Exposure to high concentrations of Manganese (Mn) is known to potentially induce an accumulation in the brain, leading to a Parkinson related disease, called manganism. Versatile mechanisms of Mn-induced brain injury are discussed, with inactivation of mitochondrial defense against oxidative stress being a major one. So far, studies indicate that the main Mn-species entering the brain are low molecular mass (LMM) compounds such as Mn-citrate. Applying a single low dose MnCl2 injection in rats, we observed alterations in Mn-species pattern within the brain by analysis of aqueous brain extracts by size-exclusion chromatography—inductively coupled plasma mass spectrometry (SEC-ICP-MS). Additionally, electrospray ionization—ion cyclotron resonance-Fourier transform-mass spectrometry (ESI-ICR/FT-MS) measurement of methanolic brain extracts revealed a comprehensive analysis of changes in brain metabolisms after the single MnCl2 injection. Major alterations were observed for amino acid, fatty acid, glutathione, glucose and purine/pyrimidine metabolism. The power of this metabolomic approach is the broad and detailed overview of affected brain metabolisms. We also correlated results from the metallomic investigations (Mn concentrations and Mn-species in brain) with the findings from metabolomics. This strategy might help to unravel the role of different Mn-species during Mn-induced alterations in brain metabolism.

An approach for manganese biomonitoring using a manganese carrier switch in serum from transferrin to citrate at slightly elevated manganese concentration

After high-dose-short-term exposure (usually from occupational exposure) and even more under low-dose long term exposure (mainly environmental) manganese (Mn) biomonitoring is still problematic since these exposure scenarios are not necessarily reflected by a significant increase of total Mn in blood or serum. Usually, Mn concentrations of exposed and unexposed persons overlap and individual differentiation is often not possible. In this paper Mn speciation on a large sample size (n=180) was used in order to be able to differentiate between highly Mn-exposed or low or unexposed individuals at low total Mn concentration in serum (Mn(S)). The whole sample set consisted of three subsets from Munich, Emilia Romagna region in Italy and from Sweden. It turned out that also at low total Mn(S) concentrations a change in major Mn carriers in serum takes place from Mn-transferrin (Mn-Tf(S)) towards Mn-citrate (Mn-Cit(S)) with high statistical significance (p<0.000002). This carrier switch from Mn-Tf(S) to Mn-Cit(S) was observed between Mn(S) concentrations of 1.5μg/L to ca. 1.7μg/L. Parallel to this carrier change, for sample donors from Munich where serum and cerebrospinal fluid were available, the concentration of Mn beyond neural barriers – analysed as Mn in cerebrospinal fluid (Mn(C)) – positively correlates to Mn-Cit(S) when Mn(S) concentration was above 1.7μg/L. The correlation between Mn-Cit(S) and Mn(C) reflects the facilitated Mn transport through neural barrier by means of Mn-citrate. Regional differences in switch points from Mn-Tf(S) to Mn-Cit(S) were observed for the three sample subsets. It is currently unknown whether these differences are due to differences in location, occupation, health status or other aspects. Based on our results, Mn-Cit(S) determination was considered as a potential means for estimating the Mn load in brain and CSF, i.e., it could be used as a biomarker for Mn beyond neural barrier. For a simpler Mn-Cit(S) determination than size exclusion chromatography inductively coupled plasma mass spectrometry (SEC-ICP-MS), ultrafiltration (UF) of serum samples was tested for suitability, the latter possibly being a preferred choice for routine occupational medicine laboratories. Our results revealed that UF could be an alternative if methodical prerequisites and limitations are carefully considered. These prerequisites were determined to be a thorough cleaning procedure at a minimum Mn(S) concentration >1.5μg/L, as at lower concentrations a wide scattering of the measured concentrations in comparison to the standardized SEC-ICP-MS results were observed.

Diverse Serum Manganese Species Affect Brain Metabolites Depending on Exposure Conditions

Occupational and environmental exposure to increased concentrations of manganese (Mn) can lead to an accumulation of this element in the brain. The consequence is an irreversible damage of dopaminergic neurons leading to a disease called manganism with a clinical presentation similar to the one observed in Parkinson's disease. Human as well as animal studies indicate that Mn is mainly bound to low molecular mass (LMM) compounds such as Mn-citrate when crossing neural barriers. The shift toward LMM compounds might already take place in serum due to elevated Mn concentrations in the body. In this study, we investigated Mn-species pattern in serum in two different animal models by size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS). A subchronic feeding of rats with elevated levels of Mn led to an increase in LMM compounds, mainly Mn-citrate and Mn bound to amino acids. In addition, a single i.v. injection of Mn showed an increase in Mn-transferrin and Mn bound to amino acids 1 h after injection, while species values were more or less rebalanced 4 days after the injection. Results from Mn-speciation were correlated to the brain metabolome determined by means of electrospray ionization ion cyclotron resonance Fourier transform mass spectrometry (ESI-ICR/FT-MS). The powerful combination of Mn-speciation in serum with metabolomics of the brain underlined the need for Mn-speciation in exposure scenarios instead of the determination of whole Mn concentrations in blood. The progress of Mn-induced neuronal injury might therefore be assessed on the basis of known serum Mn-species.

Characterization of selenium-enriched mycelia of Catathelasma ventricosum and their antihyperglycemic and antioxidant properties.

This is the first report concerning the selenium enrichment of Catathelasma ventricosum mycelia. The selenium-containing proteins present in selenium-enriched mycelia (Se-MC) were identified using size-exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS). The selenium-containing amino acids liberated by hydrolysis of these proteins were identified using anion exchange-ICP-MS. Se-MC was found to contain selenoproteins with molecular weights ranging from 1.7 to 60.5 kDa. The main selenium-containing amino acids within them were selenomethionine and selenocysteine. Furthermore, Se-MC possessed excellent antihyperglycemic and antioxidant properties. Se-MC normalized biochemical parameters like insulin level, blood glucose level, body weight, and antioxidant enzyme activity in streptozocin-induced diabetic mice. It also inhibited the α-amylase and α-glucosidase activities present in in vitro gastric and intestinal models. In conclusion, Se-MC has the potential to serve as a dietary supplement of selenium, an antioxidant, or an ingredient for the formulation of nutraceuticals.

Selectivity and specificity of small molecule fluorescent dyes/probes used for the detection of Zn2+and Ca2+in cells

Fluorescent dyes are widely used in the detection of labile (free or exchangeable) Zn(2+) and Ca(2+) in living cells. However, their specificity over other cations and selectivity for detection of labile vs. protein-bound metal in cells remains unclear. We characterized these important properties for commonly used Zn(2+) and Ca(2+) dyes in a cellular environment. By tracing the fluorescence emission signal along with UV-Vis and size exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS) in tandem, we demonstrated that among the dyes used for Zn(2+), Zinpyr-1 fluoresces in the low molecular mass (LMM) region containing labile Zn(2+), but also fluoresces in different molecular mass regions where zinc ion is detected. However, FluoZin™-3 AM, Newport Green™ DCF and Zinquin ethyl ester display weak fluorescence, lack of metal specificity and respond strongly in the high molecular mass (HMM) region. Four Ca(2+) dyes were studied in an unperturbed cellular environment, and two of these were tested for binding behavior under an intracellular Ca(2+) release stimulus. A majority of Ca(2+) was in the labile form as tested by SEC-ICP-MS, but the fluorescence from Calcium Green-1™ AM, Oregon Green® 488 BAPTA-1, Fura red™ AM and Fluo-4 NW dyes in cells did not correspond to free Ca(2+) detection. Instead, the dyes showed non-specific fluorescence in the mid- and high-molecular mass regions containing Zn, Fe and Cu. Proteomic analysis of one of the commonly seen fluorescing regions showed the possibility for some dyes to recognize Zn and Cu bound to metallothionein 2. These studies indicate that Zn(2+) and Ca(2+) binding dyes manifest fluorescence responses that are not unique to recognition of labile metals and bind other metals, leading to suboptimal specificity and selectivity.

Profiling the iron, copper and zinc content in primary neuron and astrocyte cultures by rapid online quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry

Metals often determine the chemical reactivity of the proteins to which they are bound. Each cell in the body tightly maintains a unique metalloproteomic profile, mostly dependent on function. This paper describes an analytical online flow injection quantitative size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) method, which was applied to profiling the metal-binding proteins found in primary cultures of neurons and astrocytes. This method can be conducted using similar amounts of sample to those used for Western blotting (20-150 μg protein), and has a turnaround time of <15 minutes. Metalloprotein standards for Fe (as ferritin), Cu and Zn (as superoxide dismutase-1) were used to construct multi-point calibration curves for online quantification of metalloproteins by SEC-ICP-MS. Homogenates of primary neuron and astrocyte cultures were analysed by SEC-ICP-MS. Online quantification by external calibration with metalloprotein standards determined the mass of metal eluting from the column relative to time (as pg s(-1)). Total on-column Fe, Cu and Zn detection limits ranged from 0.825 ± 0.005 ng to 13.6 ± 0.7 pg. Neurons and astrocytes exhibited distinct metalloprotein profiles, featuring both ubiquitous and unique metalloprotein species. Separation and detection by SEC-ICP-MS allows appraisal of these metalloproteins in their native state, and online quantification was achieved using this relatively simple external calibration process.

Kinetic effects on the interactions of Rh(III) with humic acids as determined using size-exclusion chromatography (SEC)

The anthropogenic inputs of Rh in the environment-together with other platinum group elements-have increased considerably during the last 20-30 years. However, thermodynamics and kinetics on the interaction of Rh with natural organic and inorganic ligands are still poorly characterized. Here, we report the time-dependent speciation of rhodium chlorides spiked to model freshwater with and without the presence of humic substances. Rhodium species were determined using size-exclusion chromatography-inductively coupled plasma mass spectrometry (SEC-ICP-MS). Results indicate that organic matter can effectively bind rhodium, but the extremely slow reaction kinetics of Rh plays a significant role on its speciation in natural waters. Accordingly, formation of Rh-organic complexes from spiked rhodium chlorides required around 15 days to reach equilibrium; this should be taken into account in those laboratory experiments where the biological interactions of Rh, using spiked samples, are studied. Regarding Rh inorganic speciation in freshwater, the available thermodynamic constants predict the dominance of the neutral trihydroxo and negatively charged tetrahydroxo rhodium complexes over typical pHs (6-8); our results, however, indicate only the presence of negatively charged hydroxocomplexes at pH 7. Reexamination of the Rh stability constants suggest that these hydroxylated rhodium complexes may also dominate its inorganic speciation in seawater.

Selenium speciation in different organs of African catfish (Clarias gariepinus) enriched through a selenium-enriched garlic based diet

Speciation of Se in fish is needed to elucidate the metabolism of this element in living organisms in the marine environment. In this paper, selenium concentration and its species distribution in several organs and tissues (liver, gills, kidney, muscle and gastrointestinal tract) of African catfish fed with a selenium-enriched garlic based diet was studied. The intention of this paper is focused on both the investigation of selenium distribution in the soluble protein fraction and the detection of selenoaminoacids. Thus, two different procedures have been developed. In the first procedure, screening of selenium in proteins in the Tris-buffer soluble fraction of different tissues was carried out by size exclusion chromatography-inductively coupled plasma-mass spectrometry (SEC-ICP-MS) and by laser ablation-inductively coupled plasma-mass spectrometry (LA-ICP-MS) after sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separation and electroblotting onto membranes. For the amino acid analysis, several sample treatments for Se-species extraction, based on enzymatic hydrolysis, were compared. The best results were obtained for incubation at controlled temperature. Application of several sample treatments in conjunction with different chromatographic techniques (reverse phase, anion exchange and ion exchange/size exclusion) was crucial to unambiguous Se-species identification. In Se-enriched African catfish a noticeable increase in the content of selenium in different organs was observed, except for the liver, where the Se level remained unaltered. The kidney was the Se-target organ in animals fed with enriched Se food. Selenomethionine (SeMet) was the main Se species identified in fillet extracts, whereas the presence of selenocysteine (SeCys) was detected in the liver and both SeMet and SeCys were present in the kidney.