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- Cis-ZEN (1)
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Eingeladener Vortrag
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Immuno imaging by the use of Laser Ablation Inductively Coupled Mass Spectrometry (LA-ICP-MS) is a growing research field in life sciences such as biology and biomedicine. Various element labeling strategies for antibodies have been developed for the application of multiplex immunoassays analyzed by the use of LA-ICP-MS. High multiplexing capabilities, a wide linear dynamic range and the possibility of absolute quantification are the main advantages of ICP-MS. But in the context of immuno imaging by the use of LA-ICP-MS, quantification of analytes is limited due to non-controllable antibody labeling chemistry. In the presented proof-of-principle a novel antibody labeling technique has been investigated which results in a controlled labeling degree. A small affinity protein based on the C2 domain of protein G was modified with conventional metal coded tags (MeCAT) after introducing a cysteine into the C-terminus of the protein. The modified C2 domain photo-crosslinks to the Fc or Fab region of the IgG and allows specific and covalent labeling of antibodies for multiplex immunoassay analysis by the use of LA-ICP-MS. In combination with a house-made calibration membrane the amount of labeled antibody–antigen complexes in a multiplex western blot immunoassay was determined by LA-ICP-MS.
A simple metal staining procedure for identification and visualization of single cells by LA-ICP-MS
(2017)
High lateral resolution of metal detection in single cells by use of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) demands powerful staining methods. In this work different staining procedures for the single cell analysis with LA-ICP-MS were optimized. An iridium intercalator was utilized to stain the cell nuclei whereas the whole cell was stained by the use of maleimido-mono-amide-DOTA (mDOTA) complexing lanthanide(III) ions. The content of the artificially introduced metals per cell was quantified using a matrix matched calibration approach based on cellulose membranes onto which standards were spotted by a microarray spotter. Absolute metal stain amounts in the range of 2.34 to 9.81 femtomole per cell were determined. The metal staining procedures allow direct identification and visualization of single cells and their cell compartments by element microscopy without the use of bright field images of the sample.
Actual research demonstrates that LA-ICP-MS is capable of being used as an imaging tool with cellular resolution. The aim of this investigation was the method development for LA-ICP-MS to extend the versatility to quantitative and multiplexing imaging of single eukaryotic cells. For visualization of individual cells selected, lanthanide-labeled antibodies were optimized for immuno-imaging of single cells with LA-ICP-MS. The molar content of the artificial introduced labels per cell was quantified using self-made nitrocellulose-coated slides for matrix-matched calibration and calculated amounts were in the range of 3.1 to 17.8 atmol per cell. Furthermore, the quantification strategy allows a conversion of 2D intensity profiles based on counts per second (cps) to quantitative 2D profiles representing the molar amount of the artificial introduced elemental probes per pixel for each individual cell.
The present study investigates the in vitro phase I metabolism of cis-zearalenone (cis-ZEN) in rat liver microsomes and human liver microsomes. cis-ZEN is an often ignored isomer of the trans-configured Fusarium mycotoxin zearalenone (trans-ZEN). Upon the influence of (UV-) light, trans-ZEN isomerizes to cis-ZEN. Therefore, cis-ZEN is also present in food and feed. The aim of our study was to evaluate the in vitro phase I metabolism of cis-ZEN in comparison to that of trans-ZEN. As a result, an extensive metabolization of cis-ZEN is observed for rat and human liver microsomes as analyzed by HPLC-MS/MS and high-resolution MS. Kinetic investigations based on the substrate depletion approach showed no significant difference in rate constants and half-lives for cis- and trans-ZEN in rat microsomes. In contrast, cis-ZEN was depleted about 1.4-fold faster than trans-ZEN in human microsomes. The metabolite pattern of cis-ZEN revealed a total of 10 phase I metabolites. Its reduction products, α- and β-cis-zearalenol (α- and β-cis-ZEL), were found as metabolites in both species, with α-cis-ZEL being a major metabolite in rat liver microsomes. Both compounds were identified by co-chromatography with synthesized authentic standards. A further major metabolite in rat microsomes was monohydroxylated cis-ZEN. In human microsomes, monohydroxylated cis-ZEN is the single dominant peak of the metabolite profile. Our study discloses three metabolic pathways for cis-ZEN: reduction of the keto-group, monohydroxylation, and a combination of both. Because these routes have been reported for trans-ZEN, we conclude that the phase I metabolism of cis-ZEN is essentially similar to that of its trans isomer. As trans-ZEN is prone to metabolic activation, leading to the formation of more estrogenic metabolites, the novel metabolites of cis-ZEN reported in this study, in particular α-cis-ZEL, might also show higher estrogenicity.
Biological systems exhibit a very high complexity, because they consist of various cell populations showing heterogenic characteristics. Therefore the individual analysis of single cells is important to understand cellular processes as well as their function in a cell system. Especially investigation in the question about what is biological variability and what is a substantial difference between two cells of the same type (which might lead to dysfunction or disease) is of high interest.
Today improved spatial and temporal resolution enable the use of laser ablation inductively coupled mass spectrometry (LA-ICP-MS) for element microscopy of single cells. Next to the characterization of natural trace elements the introduction of artificial metal labels into cells is of high interest. Element labeling of cell compartments and/or proteins allows its simultaneous analysis and localization within a cell via element microscopy. Two fast and simple metal staining procedures of adherent cells for identification of single cells via LA-ICP-MS at sub micrometer resolution are presented.
Two labeling reagents were selected which were already described in literature for applications around protein and cell analysis. An Iridium-intercalator is utilized to stain the cell nuclei whereas the whole cell is stained by maleimido-mono-amide-DOTA-complexes (mDOTA) loaded with lanthanide(III) isotope ions.
The metal staining procedures allow the visualization of single cells by element microscopy independent of a superposition of analyte’s 2D element intensity profile with a prior taken bright field image of the sample. The applicability was shown on an actual question in nano-toxicology (the cellular uptake of nanoparticles).
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.
Biological systems exhibit a very high complexity, because they consist of various cell populations showing heterogenic characteristics. Therefore the individual analysis of single cells is important to understand cellular processes as well as their function in a cell system. Especially investigation in the question about what is biological variability and what is a substantial difference between two cells of the same type (which might lead to dysfunction or disease) is of high interest. Today improved spatial and temporal resolution enable the use of laser ablation inductively coupled mass spectrometry (LA-ICP-MS) for element microscopy of single cells. Next to the characterization of natural trace elements the introduction of artificial metal labels into cells is of high interest. Element labeling of cell compartments and/or proteins allows its simultaneous analysis and localization within a cell via element microscopy. Two fast and simple metal staining procedures of adherent cells for identification of single cells via LA-ICP-MS at sub micrometer resolution are presented. An Iridium-intercalator is utilized to stain the cell nuclei whereas the whole cell is stained by maleimido-mono-amide-DOTA-complexes (mDOTA) loaded with lanthanide(III) isotope ions. The metal staining procedures allow the visualization of single cells by element microscopy independent of a superposition of analyte’s 2D element intensity profile with a prior taken bright field image of the sample.
ICP-MS is a well-established analytical method which excels by high accuracy, high dynamic range and extremely low limits of detection for most metals. Furthermore ICP-MS offers a very high multi-element coverage so that many elements of the periodic table can be detected simultaneously. In this series of lectures, we want to focus on the historical developments, fundamentals, instrumentation and novel applications of ICP-MS in the life and material sciences.