Norbert Jakubowski

Quantitative Imaging of Gold and Silver Nanoparticles in Single Eukaryotic Cells by Laser Ablation ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was utilized for spatially resolved bioimaging of the distribution of silver and gold nanoparticles in individual fibroblast cells upon different incubation experiments. High spatial resolution was achieved by optimization of scan speed, ablation frequency, and laser energy. Nanoparticles are visualized with respect to cellular substructures and are found to accumulate in the perinuclear region with increasing incubation time. On the basis of matrix-matched calibration, we developed a method for quantification of the number of metal nanoparticles at the single-cell level. The results provide insight into nanoparticle/cell interactions and have implications for the development of analytical methods in tissue diagnostics and therapeutics.

Elemental Bioimaging in Kidney by LA–ICP–MS As a Tool to Study Nephrotoxicity and Renal Protective Strategies in Cisplatin Therapies

A laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)-based methodology is presented for Pt, Cu, and Zn bioimaging on whole kidney 3 μm sagittal sections from rats treated with pharmacological doses of cisplatin, which were sacrificed once renal damage had taken place. Pt turned out to accumulate in the kidney cortex and corticomedullary junction, corresponding to areas where the proximal tubule S3 segments (the most sensitive cells to cisplatin nephrotoxicity) are located. This demonstrates the connection between platinum accumulation and renal damage proved by histological examination of HE-stained sections and evaluation of serum and urine biochemical parameters. Cu and Zn distribution maps revealed a significant displacement in cells by Pt, as compared to control tissues. A dramatic decrease in the Pt accumulation in the cortex was observed when cilastatin was coadministered with cisplatin, which can be related to its nephroprotective effect. Excellent imaging reproducibility, sensitivity (LOD 50 fg), and resolution (down to 8 μm) were achieved, demonstrating that LA-ICP-MS can be applied as a microscopic metal detector at cellular level in certain tissues. A simple and quick approach for the estimation of Pt tissue levels was proposed, based on tissue spiking.

Metallobiomolecules. The basis of life, the challenge of atomic spectroscopy

An outline of recent advances in metalloproteomics (structural and functional characterization of metal-binding proteins and their structural metal-binding moieties) and metallomics (characterization of the entirety of metal and metalloid species within a cell or tissue type) is presented. ICP-MS allows the specific detection of heteroelements after separation using techniques traditionally associated with biochemistry, such as gel electrophoresis or capillary chromatography. The element specificity, independence of matrix effects, and wide linear range make ICP-MS an attractive complementary technique to ESI/MALDI-MS for the analysis of sulfur-, selenium- and phosphorus-containing proteins, metal-complexes with nucleic acids, carbohydrates and proteins, and metal-tagged biomolecules in general.

State of the art report of selenium speciation in biological samples

Selenium is an essential trace element for humans and animals. It plays a putative role in the prevention of cancer and other illnesses. The increasing use of selenium in food and fodder supplementation has resulted in a need for high-performance analytical methods allowing the characterisation of the marketed products. Reliable and validated analytical methods applied in this research field are urgently needed. This review presents the state-of-the-art of systematic developments concerning different aspects of qualitative and quantitative selenium speciation analyses in biological samples.

Quantitative determination of DNA adducts using liquid chromatography/electrospray ionization mass spectrometry and liquid chromatography/high-resolution inductively coupled plasma mass spectrometry

The quantitative determination of nucleotides from DNA modified by styrene oxide is described using a combination of inductively coupled plasma high-resolution mass spectrometry (ICP-HRMS) and electrospray ionization mass spectrometry (ESI-MS), both interfaced to reversed-phase high-performance liquid chromatography (HPLC). LC/ICP-MS (resolution > 1500 to discriminate against 15N16O+ and 14N16OH+) was employed to determine quantitatively the content of modified nucleotides in standard solutions based on the signal of phosphorus; phosphoric acid served as an internal standard. By means of the standard addition technique the sensitivity of the LC/ESI-MS approach was subsequently determined. Since a comparison of UV, ICP and ESI-MS data suggested that in ESI-MS the ionization efficiency of the adducts is identical within the error limits, quantitative determination of all adducts is possible. For LC/ESI-MS with single ion monitoring, the detection limit for styrene oxide adducts of nucleotides was determined to be 20 pg absolute or 14 modified in 10(8) unmodified nucleotides in a 5 micrograms DNA sample, which comes close to the best methods available for the detection of chemical modifications in DNA.

Speciation of Chromium by Direct Coupling of Ion Exchange Chromatography With Inductively Coupled Plasma Mass Spectrometry

Ion exchange chromatography has been coupled with inductively coupled plasma mass spectrometry (ICP-MS) for the element speciation of chromium. Hydraulic high pressure nebulization was used for sample introduction with high aerosol efficiency. It was the particular aim of this work to exclusively use nitric acid for elution in order to reduce interferences in ICP-MS. Therefore, an anion exchange pre-column IonPac-AG5 (Dionex, Idstein, Germany) with a length of 50 mm and an id of 4 mm has been operated as a mixed-mode column, which shows cation exchange capabilities too. The column was operated at a flow rate of 1.2 ml min–1 and samples were injected by use of a sample loop with a volume of 100 µl. Both CrVI anions and CrIII cations could be retained on the column, and optimum results concerning chromatographic and mass spectrometric conditions were finally achieved with discontinuous elution in two steps by injection of 0.3 m (pH 0.5) HNO3 for CrVI and subsequently of 1.0 m (pH 0) HNO3 for CrIII. Detection limits just above 0.1 µg l–1 could be realized.

Speciation of chromium using reversed phase-high performance liquid chromatography coupled to different spectrometric detection methods

Speciation of Cr(III) and Cr(VI) based on the formation of different complexes with ammonium-pyrrolidinedithioate (APDC) in a continuous flow technique and their preconcentration using solid phase extraction (SPE) have been elaborated and applied to the analysis of waste waters from the galvanic industry. The Cr complexes were separated and determined using reversed phase-high performance liquid chromatography (RP-HPLC) coupled to different detection methods, namely UV-detection, graphite furnace-atomic absorption spectrometry (GF-AAS) and inductively coupled plasma mass spectrometry with hydraulic high pressure nebulization (HHPN/ICP-MS). After optimization the detection limits for Cr(III) and Cr(VI) of all methods are at the μg 1−1 level and the precision in terms of RSD is 5% (cCr = 100 μg 1−1, N = 10). The procedure was applied to the determination of Cr(III) and Cr(VI) at the μg 1−1 level in galvanic waste waters, and its accuracy was approved by comparing the results with those of independent methods.

Performance characteristics of inductively coupled plasma mass spectrometry with high mass resolution

A laboratory prototype is described of a new ICP-MS system with high mass resolution, based on a double focusing magnetic field analyser, which has been especially designed for application in elemental analysis. The performance characteristics of this instrument are summarized comparing operation in a low resolution mode (R= 300) and a high resolution mode (R= 3000) with reference to common quadrupole instruments with (nearly) unit resolution. Most of the typical spectral interferences can be resolved. Detection limits were achieved of one order of magnitude and more below those obtained with quadrupole instruments. Several operational features of the instrument are described.

Labelling of proteins with 2-(4-isothiocyanatobenzyl)-1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid and lanthanides and detection by ICP-MS

The labelling of two different proteins (bovine serum albumin, hen egg white lysozyme) with the commercially available chelating compound 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (p-SCN-Bn-DOTA) has been investigated. The assay described here has been optimised for the application to detect proteins labelled by stable isotopes of Eu, Tb and Ho and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Detection has been performed by laser ablation ICP-MS after electroblotting of the target proteins onto NC membranes. A range of total protein amounts from 0.015 pmol (BSA) to 105 pmol (lysozyme) has been covered. A calibration was performed for BSA in the range from 0.015 to 15 pmol and a limit of detection below 15 fmol can be estimated. For lysozyme integrated sensitivities of more than 107 cps pmol−1 of protein have been realized. The conditions, once optimised for labelling with Eu, have been applied for other lanthanides (Tb, Ho), too. ESI-MS of the intact and the tryptic digested lysozyme has been used as to provide a better understanding of the reaction chemistry and efficiency. The procedure described here looks promising to develop multielement labelling strategies (multiplexing) for ICP-MS applications in quantitative proteomics.

Multiplexed Immunohistochemical Detection of Tumor Markers in Breast Cancer Tissue Using Laser Ablation Inductively Coupled Plasma Mass Spectrometry

We optimized multiplexed immunohistochemistry (IHC) on breast cancer tissue. Up to 20 tumor markers are routinely evaluated for one patient, and thus, a common analysis results in a series of time consuming staining procedures. As an alternative, we used lanthanides for labeling of primary antibodies, which are applied in IHC. Laser ablation (LA) ICPMS was elaborated as a detection tool for multiplexed IHC of tissue sections. In this study, we optimized sample preparation steps and LA ICPMS parameters to achieve a sufficient signal-to-background ratio. The results prove the high selectivity of applied antibodies, which was sustained after labeling. Up to three tumor markers (Her 2, CK 7, and MUC 1) were detected simultaneously in a single multiplex analysis of a 5 μm thin breast cancer tissue at a laser spot size of 200 μm. Furthermore, the LA ICPMS results indicate a significantly higher expression level of MUC 1 compared to Her 2 and CK 7, which was not obvious from the conventionally stained tissue sections.