Carola Pickhardt

Copper, zinc, phosphorus and sulfur distribution in thin section of rat brain tissues measured by laser ablation inductively coupled plasma mass spectrometry: possibility for small-size tumor analysis

A microanalytical method using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was developed to measure element distribution in rat brain tissues for the detection of a small-size tumor. The stereotaxically guided tumor was implanted by injecting 5 μl of 103 F98 cells into the right Caudatus putamen of a male F344 Fisher rat brain hemisphere. The second non-treated rat brain hemisphere is used as control tissue. Tumor investigation of adjacent slices is carried out by LA-ICP-MS and, in addition, autoradiographically with a tritiated ligand (3H-PK11195) of the peripheral benzodiazepine-receptor, which is not expressed in the brain under normal, physiological conditions but during tumor development. Ion intensities of 63Cu+, 64Zn+, 31P+ and 32S+ in the rat brain section (thickness: 20 μm; analyzed area 12 mm by 6 mm) containing the local tumor and control area were measured by scanning with a focused laser beam at wavelength 213 nm, diameter of laser crater 50 μm and laser power density 3·109 W cm−2, in a cooled laser ablation chamber coupled to a double-focusing sector field ICP-MS. The quantitative determination of element distribution in a thin slice of the rat brain tissue was carried out using matrix-matched laboratory standards. The mass spectrometric analysis yielded an inhomogeneous distribution for Cu, Zn, S and P in the analyzed rat brain sections. For Cu and Zn a deficiency in and around the tumor region in comparison with the control brain tissue of the second hemisphere was found. The detection limits for distribution analysis of Zn and Cu measured by LA-ICP-MS are in the ng g−1 range. The capability and the limits of LA-ICP-MS will be studied for the imaging of element distribution in thin cross sections of brain tissues in order to create a new diagnostic method for the borders of small-size tumors.

Determination of phosphorus and metals in human brain proteins after isolation by gel electrophoresis by laser ablation inductively coupled plasma source mass spectrometry

Phosphorus, sulfur, silicon and metal concentrations (Al, Cu and Zn) were determined in human brain proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) after separation of protein mixtures by two dimensional (2-D) gel electrophoresis. The analysis of phosphorus, silicon and metals in single protein spots in the gel was performed with an optimized microanalytical method using a double-focusing sector field inductively coupled plasma mass spectrometer coupled to a commercial laser ablation system (LA-ICP-MS). Relative ion intensities for P, Si and metals with respect to sulfur in protein spots were determined by LA-ICP-MS. The detection limits for phosphorus and sulfur in protein spots with a silver staining procedure on the 2-D gels were compared with the Coomassie staining technique described previously.

Ultratrace determination of uranium and plutonium by nano-volume flow injection double-focusing sector field inductively coupled plasma mass spectrometry (nFI–ICP-SFMS)

A sensitive analytical procedure based on nano-volume flow injection (FI) and inductively coupled plasma double-focusing sector field mass spectrometry (ICP-SFMS) was developed for the ultratrace determination of uranium and plutonium. A 54-nl sample was injected by means of a nanovolume injector into a continuous flow of carrier liquid at 7 μl min−1 prior to ICP-SFMS. The absolute detection limits were 9.1 × 10−17 g (3.8 × 10−19 mol, ∼230000 238U atoms) and 1.5 × 10−17 g (6 × 10−20 mol, ∼38000 242Pu atoms) for uranium and plutonium, respectively. The method was validated for the determination of the uranium isotope ratios by the analysis of a certified isotope reference material (NIST U350). The analysis of a contaminated urine sample showed the enriched uranium to be the origin of contamination. Another application concerned the determination of plutonium at the subfemtomolar level in water with a detection limit down to the ag ml−1 range (6 × 10−18 g ml−1).

Structural identification and quantification of protein phosphorylations after gel electrophoretic separation using Fourier transform ion cyclotron resonance mass spectrometry and laser ablation inductively coupled plasma mass spectrometry

Abstract In the present work mass spectrometric approaches are described for the identification of phosphorylated protein structures, and the direct quantification of protein–phosphorus contents, using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). An ultrahigh resolution FT-ICR-MS method was developed and applied for the structural identification of phosphorylations in proteins, using direct peptide mapping analysis with high mass accuracy of tryptic phosphorylated fragments. The application of this method to human tau proteins, one of the key proteins for the formation of neurofibrillary tangles in Alzheimer’s disease, provided the identification of 17 phosphorylation sites. A high-sensitivity inorganic mass spectrometric technique has been developed for the direct determination of phosphor and sulfur concentrations in proteins separated by two-dimensional (2D) gel electrophoresis. Quantitative P and S determination in protein gel spots was performed with an optimized method using a double-focusing sector field ICP mass spectrometer coupled to a laser ablation chamber (LA-ICP-MS). Two different quantification strategies were applied: (i) determination of P and S in gel spots by LA-ICP-MS, following the determination of these elements in blank gel after trypsin and HNO 3 digestion using ICP-SFMS; (ii) a new quantification procedure by LA-ICP-MS was developed for the direct microlocal analysis in small protein spots from 2D gels. A solution-based calibration strategy in LA-ICP-MS was proposed for the quantification procedure using an ultrasonic nebulizer for introduction of calibration standard solutions coupled to the laser ablation chamber. Cobalt was used as an internal standard element, and was added to the gel at a defined concentration. The quality of phosphor determination by LA-ICP-MS was ascertained with β-casein as reference material. In a first application to the multi-phosphorylated tau protein, an average phosphorus content of ca. 20% was determined. The present results demonstrate the analytical merit of the combination of high resolution FT-ICR-MS and LA-ICP-MS for the molecular characterization of phosphorylated protein structures and determination of phosphorus and sulfur from 2D gels.

In-gel screening of phosphorus and copper, zinc and iron in proteins of yeast mitochondria by LA-ICP-MS and identification of phosphorylated protein structures by MALDI-FT-ICR-MS after separation with two-dimensional gel electrophoresis

A new screening technique using two-dimensional gels was developed in order to rapidly identify various elements in well-separated protein spots. Yeast mitochondrial proteins were separated using two-dimensional gel electrophoresis (blue native/SDS 2D-PAGE) and marked by silver staining. The 2D gels were systematically analyzed by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using a double-focusing sector field instrument. From more than 60 mitochondrial protein spots in two-dimensional gels, phosphorus, sulfur and selected metals (Cu, Zn and Fe) were detected in a short analysis time by screening 2D gel with LA-ICP-MS using a focused laser beam. In selected protein spots a quantitative element determination was performed. Ion intensities of phosphorus and metals in single protein spots in the gels were measured at medium mass resolution using an optimized microanalytical method by LA-ICP-MS and in a solution of the gel (blank) after HNO3 digestion by ICP-MS. For quantification purposes sulfur was used as the internal standard element. The detection limits for phosphorus, sulfur, copper, zinc and iron in protein spots, determined in the gel blank (Coomassie staining), were 0.18 µg g−1, 1.3 mg g−1, 6.4 µg g−1, 17.6 µg g−1 and 9.5 µg g−1, respectively. In silver staining gel a detection limit for sulfur of 137 µg g−1 was measured. Matrix-assisted laser desorption ionization Fourier transform ion cyclotron resonance mass spectrometry (MALDI-FTICR-MS) was applied for structure analysis and determination of phosphorylation sites of phosphorylated proteins. Results of the structure analysis of separated mitochondrial proteins obtained by MALDI-FTICR-MS were combined with those of the direct determination of phosphorus, sulfur and metal concentrations in protein spots in two-dimensional gels with LA-ICP-MS.

Laser ablation inductively coupled plasma mass spectrometry for the trace, ultratrace and isotope analysis of long-lived radionuclides in solid samples

Abstract The capability of laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) for determination of long-lived radionuclides in different materials (e.g., in geological samples, high-purity graphite and nonconducting concrete matrix) was investigated. The main problem in the quantification of the analytical results of long-lived radionuclides is that (except for geological samples) no suitable standard reference materials are available. Therefore, synthetic laboratory standards (graphite and concrete matrix doped with long-lived radionuclides, such as 99Tc, 232Th, 233U, 235U, 237Np, 238U) were prepared and used for quantification purposes in LA-ICP-MS. Different calibration procedures—the correction of analytical results with experimentally determined relative sensitivity coefficients (RSCs), the use of calibration curves and solution calibration by coupling LA-ICP-MS with an ultrasonic nebulizer—were applied for the determination of long-lived radionuclides, especially for Th and U in different solid samples. The limits of detection of long-lived radionuclides investigated in concrete matrix are determined in the pg g−1 range (e.g., for 237Np-50 pg g−1 in quadrupole LA-ICP-MS; for 233U-1.3 pg g−1 in double-focusing sector field LA-ICP-MS). Results of isotope ratio measurements of Th and U in synthetic laboratory standards and different solid radioactive waste materials of direct analysis on solid samples using LA-ICP-MS are comparable to measurements using the double-focusing sector field ICP-MS after separation of the analyte, even if no possible interference of atomic ions of analyte and molecular ions are expected. Furthermore, LA-ICP-MS allows precise and accurate isotope ratio measurements of Th and U in solid samples. For example, the isotope ratio 234U/238U = 0.000067 in radioactive reactor graphite was determined with a precision of 1.1% relative standard deviation (RSD).

Migration history of North Sea houting (Coregonus oxyrinchus L.) caught in Lake IJsselmeer (The Netherlands) inferred from scale transects of 88 Sr: 44 Ca ratios

Abstract.North Sea houting, Coregonus oxyrinchus, became extinct in the River Rhine in the 1940 s and was reintroduced in the 1990 s. To study the migration history of individuals, the 88Sr:44Ca ratio of scales of 39 houting (10–44 cm TL) caught in Lake IJsselmeer was analysed using laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS). Scales of houting inhabiting freshwater ponds and two Danish rivers containing the last original populations in the North Sea basin were used as controls. Fish that lived exclusively in freshwater had 88Sr:44Ca ratios of around 0.2 from the nucleus to the edge; 29 of the analysed houting from Lake IJsselmeer were of this type. Most of these were small, but some were mature and up to 42 cm in length. Seven houting had 88Sr:44Ca ratios over 0.27 from the nucleus to the scale maximum values, indicating migration to the sea at early life stages. Three houting with low 88Sr:44Ca ratios at the scale nucleus and increased 88Sr:44Ca ratios towards the scale edge probably lived in freshwater for a longer period after hatching and then moved to brackish/marine environments. The scale analysis indicates different migration patterns for houting in Lake IJsselmeer and provides evidence that this species (1) is sometimes able to pass the migratory barriers between the Wadden Sea and Lake IJsselmeer, and (2) does not need to migrate to sea to reach maturity.