Miroslav Zoriy

Bioimaging of metals by laser ablation inductively coupled plasma mass spectrometry (LA‐ICP‐MS)

The distribution analysis of (essential, beneficial, or toxic) metals (e.g., Cu, Fe, Zn, Pb, and others), metalloids, and non-metals in biological tissues is of key interest in life science. Over the past few years, the development and application of several imaging mass spectrometric techniques has been rapidly growing in biology and medicine. Especially, in brain research metalloproteins are in the focus of targeted therapy approaches of neurodegenerative diseases such as Alzheimers and Parkinsons disease, or stroke, or tumor growth. Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using double-focusing sector field (LA-ICP-SFMS) or quadrupole-based mass spectrometers (LA-ICP-QMS) has been successfully applied as a powerful imaging (mapping) technique to produce quantitative images of detailed regionally specific element distributions in thin tissue sections of human or rodent brain. Imaging LA-ICP-QMS was also applied to investigate metal distributions in plant and animal sections to study, for example, the uptake and transport of nutrient and toxic elements or environmental contamination. The combination of imaging LA-ICP-MS of metals with proteomic studies using biomolecular mass spectrometry identifies metal-containing proteins and also phosphoproteins. Metal-containing proteins were imaged in a two-dimensional gel after electrophoretic separation of proteins (SDS or Blue Native PAGE). Recent progress in LA-ICP-MS imaging as a stand-alone technique and in combination with MALDI/ESI-MS for selected life science applications is summarized.

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) in elemental imaging of biological tissues and in proteomics

Of all the inorganic mass spectrometric techniques, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) plays a key role as a powerful and sensitive microanalytical technique, enabling multi-element trace analysis and isotope ratio measurements at the trace and ultra-trace level in the life sciences. LA-ICP-MS was used to produce images of detailed regionally specific element distribution in thin sections of tissue from different parts of the human brain. The quantitative determination of copper, zinc and other elements distributed in thin slices of human brain samples was performed using matrix-matched laboratory standards. Imaging mass spectrometry provides new information on the spatially inhomogeneous element distribution in thin sections of human tissue, for example of different brain regions (e.g., insular region) or brain tumour tissue. The detection limits obtained for Cu and Zn determination in tissue sections were in the sub-µg g–1 range. Possible strategies will be discussed for applying LA-ICP-MS in brain research and the life sciences, including the imaging of thin slices of brain tissue in order to obtain element distributions or applications in proteome analysis in combination with MALDI-MS to study phospho- and metal-containing proteins.

Quantitative imaging of zinc, copper and lead in three distinct regions of the human brain by laser ablation inductively coupled plasma mass spectrometry

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used to determine the distribution of the trace elements zinc, copper and lead in insular, central and hippocampal areas of thin tissue sections (thickness 20microm) through an entire human brain hemisphere. For the investigation of the tissue samples, a commercial laser ablation system was coupled to a double-focusing sector field ICP-MS. The regions of interest of healthy brain tissue (thickness 20microm) were scanned (raster area approximately 200mm(2)) with a focused laser beam (wavelength 266nm, diameter of laser crater 200microm and laser power density 3x10(9)Wcm(-2)). The ion intensities of (64)Zn(+), (63)Cu(+) and (208)Pb(+) were measured by LA-ICP-MS within the ablated area. For quantification purposes, matrix-matched laboratory standards were prepared by means of dosing of each analyte to the pieces of brain tissue. The mass spectrometric analysis yielded inhomogeneous and largely reciprocal distributions of Zn and Cu in the selected areas of investigated brain samples. The highest concentrations of Zn and Cu with the most distinct distribution pattern were found in the hippocampus (up to 15microg g(-1)). In contrast to zinc and copper, for lead, a more homogeneous distribution throughout all regions examined was found at a low concentration (in the ngg(-1) range) level within the analytical range of LA-ICP-MS.

Imaging of nutrient elements in the leaves of Elsholtzia splendens by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) was used for the quantitative imaging of nutrient elements (such as K, Mg, Mn, Cu, P, S and B) in the leaves of Elsholtzia splendens. The plant leaves were scanned directly with a focused Nd:YAG laser in the laser ablation chamber. The ablated material was transported with argon as carrier gas to a quadrupole-based ICP-MS (ICP-QMS), and the ion intensities of (39)K(+), (24)Mg(+), (55)Mn(+), (63)Cu(+), (31)P(+), (34)S(+) and (11)B(+) were measured by ICP-QMS to study the distribution of the elements of interest. The imaging technique using LA-ICP-MS on plant leaves does not require any sample preparation. Carbon ((13)C(+)) was used as an internal standard element to compensate for the difference in the amount of material ablated. Additional experiments were performed in order to study the influence of the water content of the analyzed leaves on the intensity signal of the analyte. For quantification purposes, standard reference material (NIST SRM 1515 Apple Leaves) was selected and doped with standard solutions of the analytes within the concentration range of 0.1-2000 mg L(-1). The synthetic laboratory standards together with the samples were measured by LA-ICP-MS. The shape and structure of the leaves was clearly given by LA-ICP-MS imaging of all the elements measured. The elemental distribution varied according to the element, but with a high content in the veins for all the elements investigated. Specifically, Cu was located uniformly in the mesophyll with a slightly higher concentration in the main vein. High ion intensity was measured for S with a high amount of this element in the veins similar to the images of the metals, whereas most of the B was detected at the tip of the leaf. With synthetic laboratory standard calibration, the concentrations of elements in the leaves measured by LA-ICP-MS were between 20 microg g(-1) for Cu and 14,000 microg g(-1) for K.

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.

Evidence of near-field laser ablation inductively coupled plasma mass spectrometry (NF-LA-ICP-MS) at nanometre scale for elemental and isotopic analysis on gels and biological samples

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) has been established as a powerful surface analytical method for local elemental analysis on metallic, ceramic, geological or biological sample surfaces. Here we show a new way of nanometre scale analysis of elements on sample surfaces by near-field LA-ICP-MS (NF-LA-ICP-MS). This technique uses the near-field enhancement effect on the tip of a thin silver needle in a laser beam (Nd:YAG laser, wavelength 532 nm) on the sample surface. The thin silver needle was etched electrolytically in an electrochemical cell using a droplet of citric acid as electrolyte. For nanolocal analysis by NF-LA-ICP-MS on soft matter (e.g., on 2-D gels and biological samples) a small volume transparent laser ablation chamber was constructed and coupled to a double-focusing sector field inductively coupled plasma mass spectrometer (ICP-MS). A small amount of soft sample material is ablated at atmospheric pressure by a single laser shot in the near-field of the silver tip in the defocused Nd:YAG laser beam. The ablated material is transported with argon as carrier gas into the inductively coupled plasma (ICP) ion source of the sensitive double-focusing sector field mass spectrometer with reverse Nier–Johnson geometry. By single-shot analysis on 2-D gels and biological surfaces doped with uranium in the μg g−1 range using NF-LA-ICP-MS an enhancement of ion intensities of transient signals in comparison with the background signal of up to factor 60 was observed. In gels doped with isotopically enriched 65Cu and 67Zn spikes by NF-LA-ICP-MS (single shot analysis) ion intensities up to the n × 105 cps range and isotope ratios (235U/238U, 65Cu/63Cu and 67Zn/64Zn) were measured at a lateral resolution in the nanometre scale. Using the near-field effect in LA-ICP-MS, it was demonstrated that nanolocal analysis is possible in single-shot measurements of elements on biological samples and on a gel surface with spatial resolution at the hundreds of nanometres range. This first experiment on near-field LA-ICP-MS opens up a new, challenging path for future applications in nanoimaging of elements in life science, biology and medicine, e.g., for analyses of single cells, cell organelles or biological structures at nanometre range in order to detect neurodegenerative diseases, but also in material science, nanotechnologies and nanoelectronics.

Analysis of metal-binding proteins separated by non-denaturating gel electrophoresis using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS)

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) and laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) have become established as very efficient and sensitive biopolymer and elemental mass spectrometric techniques for studying metal-binding proteins (metalloproteins) in life sciences. Protein complexes present in rat tissues (liver and kidney) were separated in their native state in the first dimension by blue native gel electrophoresis (BN-PAGE). Essential and toxic metals, such as zinc, copper, iron, nickel, chromium, cadmium and lead, were detected by scanning the gel bands using quadrupole LA-ICP-MS with and without collision cell as a microanalytical technique. Several proteins were identified by using MALDI-TOF-MS together with a database search. For example, on one protein band cut from the BN-PAGE gel and digested with the enzyme trypsin, two different proteins - protein FAM44B and cathepsin B precursor - were identified. By combining biomolecular and elemental mass spectrometry, it was possible to characterize and identify selected metal-binding rat liver and kidney tissue proteins.

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.

Imaging of essential and toxic elements in biological tissues by LA-ICP-MS

Laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) using double-focusing sector field (LA-ICP-SFMS) or quadrupole-based mass spectrometers (LA-ICP-QMS) was employed as powerful imaging (mapping) techniques to produce images of detailed regionally specific element distributions in thin tissue sections of different sizes such as control rat brain tissues or tumor regions to study tumor growing. LA-ICP-QMS was employed to investigate the uptake and the transport of nutrient elements in plant tissues or imaging of metals in proteins separated by native 2D BN-PAGE.

Study of formation of Cu- and Zn-containing tau protein using isotopically-enriched tracers by LA-ICP-MS and MALDI-FTICR-MS

Metal-containing proteins were detected directly in separated protein bands in one-dimensional gels, by laser ablation inductively coupled plasmamass spectrometry (LA-ICP-MS) as an atomic mass spectrometric technique. In order to study the binding of Cu and Zn on tau protein isoforms as a target protein in Alzheimer’s disease, enriched isotope tracers (65Cu and 67Zn) were doped to one-dimensional gels of separated tau protein isoforms after gel electrophoresis. In several protein bands metal ions were detected and 65Cu/63Cu and 67Zn/64Znisotope ratios were measured by LA-ICP-MS. The isotope analysis by LA-ICP-MS indicates certain proteins with a natural isotope composition of Cu or Zn. However, copper-containing tau protein isoforms with a changed isotope ratio in comparison to the isotope composition in nature were also found. This experimental finding demonstrates the formation of new metal-containing tau protein complexes during the tracer experiments in the 1-D gel. We attempted to identify the protein bands from tau proteinsseparated by one-dimensional (1-D) gel electrophoresis by using biopolymermass spectrometry with MALDI-FTICR-MS, after excision from the 1-D gel and tryptic digestion.