Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis

A method was developed for the precise and accurate determination of ovalbumin labelled with p-hydroxy-mercuribenzoic acid (pHMB) using polyacrylamide gel electrophoresis with ns-laser ablation-inductively coupled plasma mass spectrometry. Following systematic optimisation of the ablation process in terms of detection sensitivity, two different quantification strategies were applied: external calibration using standards of the derivatized protein after 13C+ normalization and, as a proof of concept, label-specific isotope dilution analysis (IDA) using pHMB enriched in the isotope 199Hg. Due to the inhomogeneous distribution of the protein within the gel bands, it could be demonstrated that the IDA approach was superior in terms of precision and accuracy. Furthermore, it permits a reliable quantification, if more complex separation protocols are applied, as typically occurring analyte loss and degradation can be compensated for as soon as complete mixture of spike and sample is achieved. The estimated limit of detection was 160fmol in the case of ovalbumin. In contrast to earlier studies using metals naturally present in proteins, no loss of mercury was observed during separation under denaturing conditions and other sample preparation steps. Using label-specific IDA, the measured isotope ratios in the gel corresponded to recoveries between 95% and 103

Tracking soluble and nanoparticulated titanium released in vivo from metal dental implant debris using (single-particle)-ICP-MS

Background: This work studies the presence of the Ti, Al and V metal ions and Ti nanoparticles released from the debris produced by the implantoplasty, a surgical procedure used in the clinic, in rat organs. Methods: The sample preparation for total Ti determination was carefully optimized using microsampling inserts to minimize the dilution during the acid attack of the lyophilized tissues by a microwave-assisted acid digestion method. An enzymatic digestion method was optimized and applied to the different tissue samples in order to extract the titanium nanoparticles for the single-particle ICP-MS analysis.Results: A statistically significant increase was found for Ti concentrations from control to experimental groups for several of the studied tissues, being and particularly significant in the case of brain and spleen. Al and V concentrations were detected in all tissues but they were not different when comparing control and experimental animals, except for V in brain. The possible presence of Ti-containing nanoparticles mobilized from the implantoplasty debris was tested using enzymatic digestions and SP-ICP-MS. The presence of Ti-containing nanoparticles was observed in all the analyzed tissues, however, differences on the Ti mass per particle were found between the blanks and the digested tissue and between control and experimental animals in some organs.Conclusion: The developed methodologies, both for ionic and nanoparticulated metal contents in rat organs, have shown the possible increase in the levels of Ti both as ions and nanoparticles in rats subjected to implantoplasty

Analysis of hepcidin, a key peptide for Fe homeostasis, via sulfur detection by capillary liquid chromatography-inductively coupled plasma mass spectrometry

Since its discovery the role of hepcidin as key regulator of iron homeostasis has been stressed by many authors. This peptide hormone of 25 amino acids, out of which 8 are cysteines, holds promise as a novel biomarker in iron metabolism disorders. In this work, we illustrate the progress of a new method for the analysis of hepcidin via sulfur detection using inductively coupled plasma mass spectrometry (ICP-MS) after capillary liquid chromatography for separation of the species. Three different ICP-MS-based strategies have been evaluated to overcome S polyatomic interferences: (1) a collision/reaction cell instrument with Xe as collision gas; (2) the monitoring of SO+ by adding O2 to the reaction cell and (3) a double focusing system (DF-ICP-MS). The latter one provided best limits of detection for S (7 ng mL−1) and good precision and accuracy to monitor S isotope ratios so it was used for hepcidin determination in urine samples by online isotope dilution. Quantitative recoveries of the peptide standard (101.7 ± 1.4%) are obtained with the proposed setup after controlling the column temperature (50 °C) and using the X-skimmer cone. Different sample clean-up procedures were studied in order to apply the developed quantitative methodology to urine samples. Multidimensional (dialysis + solid phase extraction) procedures provided best results yielding a 12-fold preconcentration factor. The obtained extracts were analyzed simultaneously by the developed capLC-ICP-MS setup and also by capLC-ESI-q-TOF for confirmation purposes. The results obtained revealed that ESI-q-TOF detection is more suitable for hepcidin determination in urine samples regarding both selectivity and sensitivity

Systematic studies on the determination of Hg-labelled proteins using laser ablation-ICPMS and isotope dilution analysis

ISSN:1618-2650ISSN:1618-264

Capabilities of Single Cell ICP-MS for the Analysis of Cell Suspensions from Solid Tissues

Single cell elemental (SC) analysis of isogenic cell cultures can be done using inductively coupled plasma (ICP-MS) detection. However, 2D cell cultures are just models to simplify the complexity of real tissue samples. Here, we show for the first time the capabilities of the technique (SC-ICP-MS) to analyze single cell suspensions of isolated cells from tissues. An optimized cocktail of proteolytic and collagenolytic enzymes was applied in a single preparation step with cellular yields up to 28% using 0.5 g of fresh rat spleen and liver, respectively. The retrieved cells revealed adequate morphology and stability to be examined by SC-ICP-MS. Quantitative elemental analysis of P, S, Cu, and Fe from disaggregated cells from rat spleen and liver tissues revealed levels of Fe of 7–16 fg/cell in the spleen and 8–12 fg/cell in the liver, while Cu was about 3–5 fg/cell in the spleen and 1.5–2.5 fg/cell in the liver. Evaluation of the transmembrane protein transferrin receptor 1 (TfR1) expression levels in disaggregated cells was also conducted by using a Nd-labelled antibody against this cell surface biomarker. Quantitative results showed significantly lower expression in the disaggregated cells than in the cell model HepG2, in agreement with the overexpression of this biomarker in tumor cells. In this proof of concept study, the tissue disaggregation protocol has shown to maintain the elemental intracellular content of cells as well as the presence of relevant antigens. This opens a completely new area of research for SC-ICP-MS in tissue samples as a complementary strategy with validation capabilities