LA-ICP-MS Advances Research into Alzheimer's and Other Neurogenerative Diseases

Dr. habil. J. Sabine Becker, head of the BrainMet (Bioimaging of Metals in Brain and Metallomics) Centre of Excellence of the Central Division of Analytical Chemistry, Research Center, Juelich, Germany, has been using laser-ablation inductively coupled plasma–mass spectrometry (LA-ICP-MS) to study metals, metalloids, and non-metals in native brain sections and metallomics studies. Spectroscopy recently spoke with her about her pioneering work in this area.

Spectroscopy: How did you become interested in studying neurodegenerative diseases?

Becker: It is well known that metal ions of Fe, Cu, Zn, and others are essential for human health, are required for the brain’s biochemistry, and protect against many diseases. On the other hand there is growing evidence that these metals may play a role in Alzheimer’s disease, Parkinson’s disease, Wilson’s disease, and other illnesses. In most neurodegenerative diseases, abnormal accumulations of metals are observed in the brain, leading to the destruction of the neuronal network and finally to cell death.

I became interested in studying neurodegenerative diseases through my daughter’s (Julia Susanne Becker) PhD work at University Konstanz. She studied 10 years earlier in Michael Przybylski’ group (Analytical Chemistry and Biopolymer Structure Analysis, Department of Chemistry) the Alzheimer’s target protein “Tau” and hyperphosphorylation by high-resolution matrix-assisted laser desorption–ionization electrospray ionization–Fourier transform ion cyclotron resonance (MALDI–ESI-FTICR) mass spectrometry. So we developed microanalytical techniques for the protein spots in 1D and 2D gels by LA–ICP–MS and detected firstly phosphorus and metals in separated proteins of Alzheimer’s brain samples. In order to detect fast heteroatoms in separated proteins, we created analytical protocols from single point via line scan measurements and 2D imaging procedures of gels and studied the formation of metalloproteins using highly enriched stable isotopes. We developed the screening techniques for proteins of 2D gels and the quantification procedure of phospho- and metalloproteins by the combination of LA-ICP-MS and MALDI–FTICR–MS.

Parallel to the new emerging metallomics research, we have been looking for metal distribution in biological tissue sections. For this purpose we developed quantitative imaging techniques of elements by LA–ICP–MS. The Analytical BrainMet (Bioimaging of Metals in Brain and Metallomics, see also www.brainmet.de ) Competence Centre at the Forschungszentrum Jülich, Germany, was opened in cooperation with Thermo Fisher Scientific in December 2009 to create novel imaging procedures for biological tissue to study the role of metals and metalloproteins in thin cryosections of brain tissue in respect to neurodegenerative diseases, aging, tumor growth, or other brain lesions.

Spectroscopy: How is ICP or ICP–MS used in that work?

Becker: We work on LA-ICP-MS for bioimaging of elements (metals, metalloids, and non-metals) in native brain sections and metallomics studies. In LA-ICP-MS the inductively coupled plasma is used for atomization and ionization of laser-ablated material. The single charged ions formed in the atmospheric pressure plasma are extracted via an interface in the high vacuum mass analyzer and separated by mass-to-charge ratio (m/z) by a quadrupole-based analyzer or by a more sensitive double-focusing sector field mass spectrometry (ICP-SFMS) system.

During collecting more than 35 years extensive experience in all fields of inorganic mass spectrometry (ICP-MS, SSMS, TIMS, rf-GDMS, SIMS, SNMS) I have developed ultrahigh vacuum laser ion sources for laser ionization MS (LIMS) and studied ion formation in laser plasmas in the beginning of eighties (also see my textbook “Inorganic Mass Spectrometry: Principles and Applications”, Wiley Dec. 2007, ISBN: 978-0-470-01200-0). Microanalytical techniques by coupling of an atmospheric pressure laser-ablation system with ICP-MS were introduced in my MS laboratory in 1993 using a homemade LA system for materials research (for example, for ultrasensitive determination of trace contaminants in high-purity materials, for surface analysis and for analysis of ceramics), for determination of long-lived radionuclides in solid samples, and later using commercial LA systems for investigation for solid state physics, for isotope ratio measurements, for geology, environmental, and forensic studies, and even for life-science applications.

The Analytical BrainMet Centre at the Forschungszentrum Jülich, Germany, has developed novel quantitative mass spectrometry imaging (MSI) techniques using LA-ICP-MS) to advance life sciences.

Spectroscopy: What is innovative about your approach to this type of analysis?

Becker: The challenges of the novel BrainMet tool by LA-ICP-MS imaging are the ability to study the inhomogeneous distribution of elements in biological samples (thin tissue cryosections), and to detect heteroatoms in proteins separated in gels.

First we investigated single protein spots separated in 2D gels in respect to metal and phosphorus content and combined LA-ICP-MS with high-resolution MALDI-MS for identification of metal-containing and phosphoproteins and their quantification. Metal-containing proteins were also imaged in a 2D gel after electrophoretic separation of proteins (sodium dodecyl sulfate [SDS] or Blue Native polyacrylamide gel electrophoresis [PAGE]).

In the following studies we created quantitative LA-ICP-MS of detailed regionally specific element distributions in thin tissue sections of human or rodent brain at spatial resolution of 10–100 µm. For quantification of analytical data matrix, matched laboratory brain standards and solution-based calibration procedures were applied.

Prior to the establishment of the BrainMet Centre, no studies had been performed on the quantitative distribution of elements combined with the analysis of metalloproteins in biological tissue.

By high spatial resolution MSI using laser-induced ICP techniques, we are able to map essential and toxic metals (Li, Na, Mg, K, Ca, Al, Ti, Mn, Fe, Cu, Zn, Ni, Mo, Gd, and other rare earth metals, Cd, Hg, Pb, Tl, Th, U, and others), metalloids (such as As, Se, and Sb) and non-metals (C, P, S, Cl, Br, I) in soft tissue sections to study the metallo-architecture within the diseased brain sample compared to controls.

Spectroscopy: What obstacles, if any, did you have to overcome?

Becker: The general problem of ICP-MS in trace, species, and isotope analysis is possible isobaric interferences. MS measurements are based on measuring the ion currents of separated ion beams of isotopes. With the knowledge of the isotopic composition of elements (isotopic pattern) and a comparison of isotope images of an element, interferences can be identified easily. However, it is possible to separate isobaric interferences by application of sector-field ICP-MS at the required mass resolution or by using collision-cell ICP-MS.

It is important to note that the scarcity of native frozen pathological and healthy human brain tissue is actually one of the major bottlenecks concerning the studies of neurodegenerative diseases. Storage of tissue specimens in formalin solution (mostly commonly 4% buffered) implicates the cross-linkage of macromolecules and a washout of soluble species. Therefore we are investigating formalin-fixed samples (and paraffin-embedded tissue) compared to native tissue by our BrainMet tools and have found wash out of elements such as Na, K, Sr, and Ca and redistribution of Zn. Our conclusions are that for imaging studies, fresh native tissue cryosections are urgently required.

Spectroscopy: What results have you seen so far?

Becker: The established MSI techniques using LA-ICP-MS with spatial resolution in the 30–60 µm range were employed to study the elemental distribution and the origin of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or Wilson’s diseased brain, epilepsy, and their effects on aging of the brain, and in brain lesions.

In addition, we are developing emerging quantitative imaging techniques of biological tissue with spatial resolution at the low-micrometer and submicrometer scale using the near field effect in laser ablation (NF-LA) and by development of online coupling of a developed laser micro dissection (LMD) system to ICP–MS.

Spectroscopy: What are your next steps in this work?

Becker: In our next steps we will focus on improvement of spatial resolution of laser-induced imaging techniques and limit of detection of elements by coupling LMD- and LA- systems with a double focusing sector field (SF) ICP–MS system. LMD-ICP-SFMS imaging will soon allow us to chemically image small tissues and cellular substructures with high spatial resolution.

In addition, other forthcoming research activities will include MALDI-MS imaging (using a high-resolution MALDI–LTQ Orbitrap MS system) of biomolecules (especially the metallo- and phosphoproteins) within the brain and the combination with LA- and LMD-ICP-MS images of diseased brain. This future BrainMet work to study neurodegenerative diseases was made possible by new instruments (MALDI–LTQ Orbitrap MS and a special LMD with a 110-µJ Nd:YAG laser for ablation of biological sample) granted from Deutsche Forschungsgemeinschaft.

I would like to acknowledge Thermo Scientific (Bremen, Germany) and Deutsche Forschungsgemeinschaft (DFG grant number BE 2649/5-1) for instrumental support of the BrainMet laboratory.