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While MALDI and ESI mass spectrometry can be applied ... Keywords: biological tissues, imaging mass spectrometry, copper, laser ablation inductively coupled ...
J.S. Becker et al., Eur. J. Mass Spectrom. 13, 1–6 (2007) 

Imaging mass spectrometry in biological tissues by laser ablation inductively coupled plasma mass spectrometry

J.S. Becker,a J.Su. Becker,b M.V. Zoriy,a J. Dobrowolskaa and A. Matuschc a

Central Division of Analytical Chemistry, Research Centre Jülich, D-52425 Jülich, Germany

b

Laboratoire de Chimie Analytique Bio-Inorganique et Environnement, Centre Technologique Hélioparc, 64053 Pau, France

c

Institute of Medicine, Research Centre Jülich, D-52425 Jülich, Germany

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 trace and ultratrace level. LA-ICP-MS ���������� was �������������������������������������������������������������������������������� used to produce images of detailed regionally-specific element distribution in 20 µm thin sections of different parts of the human brain. The quantitative determination of copper, zinc, lead and uranium distribution in thin slices of human brain samples was performed using matrix-matched laboratory standards via external calibration procedures. Imaging mass spectrometry provides new information on the spatially inhomogeneous element distribution in thin sections of human tissues, for example, of different brain regions (the ������������������������������������������������������������� insular region����������������������������������������������� ) or brain tumor tissues. The detection limits obtained for Cu, Zn, Pb and U were in the ng g–1 range. ����������������������������������������������������������������������������� Possible strategies of LA-ICP-MS in brain research and life sciences include the elemental imaging of thin slices of brain tissue or applications in proteome analysis by combination with matrix-assisted laser desorption/ionization MS to study phospho- and metal-containing proteins will be discussed. Keywords: biological tissues, imaging mass spectrometry, copper, laser ablation inductively coupled plasma mass spectrometry, MALDIFT-ICR-MS, metalloproteins, selenium, zinc

The quantitative determination of essential elements (for example, P, Cu, Fe, Zn, Mn, Co, Se and others) in biological tissues is important in brain research and life sciences, for example, for studying many ­neurodegenerative diseases. A deficiency or excess of these essential elements in proteins and in human tissue have been observed in neuro­degenerative diseases (including Alzheimer’s and Parkinson’s disease), but metals can also catalyse cytotoxic reactions and are toxic at high concentrations.1,2 In particular, phosphorylated proteins (phosphoproteins) and metal-containing proteins (metalloproteins) play an essential role as cofactors in biological systems (for example, in single cells or cell organelles) and are gaining increasing ­ attention in proteomics research. Therefore, the proteomics area requires versatile and powerful analytical techniques for the detection and characterization of phosphorus, ­selenium and metal-containing

DOI: 10.1255/ejms.833

proteins within a large pool of proteins, for example, after electrophoretic separation in two-dimensional (2D) gels.3–8 For the detection of metals, P and Se in protein spots and bands directly in one- and two-dimensional gels after electrophoretic separation, laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) is the method of choice. On the other hand, mass spectrometers that employ soft ionization techniques [electrospray ionization (ESI) and matrix assisted laser desorption/ionization (MALDI)] permit the identification of large biomolecules such as proteins.4,9 While MALDI and ESI mass spectrometry can be applied for the identification of phosphorylation sites in proteins, these techniques cannot provide direct quantitative determinations of phosphorus and metals in biological samples. Therefore, only the combination of LA-ICP-MS as an element analytical technique with a biomolecular mass spec-

ISSN 1469-0667

© IM Publications 2007



Imaging Mass Spectrometry in Medical and Biological Tissues with LA-ICP-MS

trometric technique such as high-resolution MALDI-Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) allows the molecular identification and quantification of protein phosphorylation as well as of metal concentrations. This also enables post-translational modifications of proteins, for example, phosphorylation,3,4,6,7,10 to be studied which are relevant in many fundamental cellular functions, such as survival, differentiation, structural organization and stress responses, for many pathophysiological processes in carcinogenesis or neurodegenerative diseases.4 In addition, it is well known that metals (for example, Cu, Fe, Zn, Mn, Ca, Mg and others) and non-metals, such as P and S, are inhomogeneously distributed in biological tissues. Therefore, investigating the element distribution in thin tissue slices requires sensitive analytical techniques with high spatial resolution and is required in analytical chemistry. The advantages of LA-ICP-MS compared to other surface analytical techniques, such as scanning electron microscopy with energy-­dispersive X-ray analysis (SEM-EDX), 11 ­ microproton-induced Xray emission (PIXE),12 auto­radiography13 or secondary ion mass spectrometry (SIMS),14–16 are its high sensitivity (and consequently very low detection limits) for trace element determination and its ability to quantify analytical data on medical tissues due to the significantly lower matrix effects. The application of LA-ICP-MS in life sciences and in medicine focuses, at present, on individual tasks, for example, the mapping of copper and zinc in liver sections of sheep,17 the extreme ultratrace and isotope analysis of actinides (especially of plutonium) in moss samples 18 or in body fluids (urine).19 In our recent studies, LA-ICP-MS was applied for the microlocal analysis of selected protein spots in gels after two-dimensional (2D) gel electrophoresis and also for the determination of elemental distribution in thin sections of brain tissue (imaging).4,10,20 Possible strategies of LA-ICP-MS in brain research and life sciences are summarized in Figure 1. They include the imaging of thin slices of brain tissue in order to obtain images of elements (left-hand side of Figure 1) or application in proteome analysis. The combination of both approaches is a future task. LA-ICP-MS was developed as a microanalytical method in our laboratory for the determination of P, S, Si and metal concentrations (Al, Zn, Cu und Fe) in well-separated protein spots after two­dimensional gel electrophoresis in brain samples of patients with Alzheimer’s and in control brains,21 where a combination of atomic and molecular mass spectrometric methods (LA-ICP-MS and MALDI-FT-ICR-MS, respectively) was helpful for the characterization and identification of several human Alzheimer brain proteins.3,21 For these studies, a brain protein mixture was separated by two-dimensional (2D) gel electrophoresis (see 2D gel in Figure 1, top right) and the protein spots were fast-screened by microlocal analysis using LA-ICP-MS with respect to P, S, Cu, Zn and Fe content. To solve the interference problem arising in LA-ICP-MS of isobaric interference on atomic ions of analyte by the formation of molecular ions such as

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N16O+, 14N17O+ and 14N16O1H+ at mass 31 u for 31P+ or 32S2+ during 64Zn+ determination, respectively, a double-focusing sector field mass spectrometer was applied at mass resolution (m / ∆m ∼ 4400) for the analysis of small protein samples or during imaging analysis in brain tissues. In Figure 2(a), the transient signals of 31P+, 63Cu+, 56Fe+ and 64Zn+ are shown in protein spots 6–9 compared to the background signal of the blank gel. Selected protein spots in 2D gel containing these elements were investigated after tryptic digestion by MALDI-FT-ICR-MS. Results of the structure analysis of human brain proteins by MALDI-FT-ICR-MS were combined with those of the direct determination of phosphorus, copper, zinc and iron concentrations in protein spots with LA-ICP-MS. Subsequently, the element concentrations (P, Cu, Zn and Fe) were determined in the identified human brain proteins by LA-ICP-MS in 2D gel (using sulfur as the internal standard element). Due to the lack of suitable standard reference material, reliable calibration strategies were developed for the direct microlocal analysis of phosphorus and metals in protein spots and in thin sections of brain tissue using LA-ICP-MS.4,20,22 For example, in our laboratory the application of a solution-based calibration strategy was proposed for quantitative phosphorus determination by LA-ICP-MS3 and the simultaneous determination of P, S, Si, Al, Cu and Zn concentrations in human brain proteins (Alzheimer’s disease) or for imaging thin sections of brain tissue.22 In a special arrangement for solution-based calibration a micro-nebulizer was inserted directly in the laser ablation chamber for quantification purposes.23 From the results of atomic and molecular mass spectrometric techniques, the human brain proteins were characterized with respect to their structure, sequence, phosphorylation state and also metal content. For example, protein spot 6 was identified as creatine kinase, β-chain [Figure 2(b), M.W. 42.91 kDa and a phosphorylation state of 1]. By two-dimensional imaging of brain tissues, a defined sample area (several cm²) of a thin section of brain tissue (thickness: 20 µm) was ablated line by line with a focused laser beam in a cooled laser ablation chamber (using two Peltier elements behind the target holder made of aluminum).24 The spot size of the laser beams was about 50 µm and the laser power density was 3 × 109 W / cm². Ion intensities of the analytes, for example, 31P+, 32S+, 63Cu+, 64 Zn+ or 208Pb+, were measured by LA-ICP-MS within the area of interest in different regions of the human brain tissue (for example, hippocampus, insular and a precentral regions). Matrix-matched homogenized laboratory standards with well-defined element concentrations of analytes were prepared and utilized for quantification of LA-ICP-MS data in the imaging of brain tissue in routine mode. 20,22,25 As a result of LA-ICP-MS measurement, layered structures for P, S, Cu, Zn and Pb in different section of brain tissues were detected in the µg g–1 concentration range. In contrast, Th and U in the low ng g–1 range were found in brain proteins and also tissues (with a more

J.S. Becker et al., Eur. J. Mass Spectrom. 13, 1–6 (2007) 

Figure 1. Strategies of LA-ICP-MS in brain research: imaging of thin sections of brain tissues and screening of 2D gel with respect to phosphoproteins and metal-containing proteins.

homogeneous distribution).22 Recently, Dobrowolska et al.25 studied the Zn, Cu and Pb distribution in a sub-region of the brain hemisphere. Figure 3 shows the distribution of Zn and Cu in the insular region of the human brain measured by LA-ICP-MS. The layered distribution pattern of both elements is clearly visible. In additional studies, copper and zinc distribution within various sub-regions of the hippocampus were first quantitatively measured.25 A fine band inside the dentate gyrus, corresponding to its granular layer, presents the highest Cu concentrations. The Cu concentration is high along the pyramidal layer of the cornu ammonis. In the hilus region, only that part of the pyramidal layer in the convexity closest to the dentate gyrus displays Cu accumulation. Zn is most highly concentrated, for example, in the dentate gyrus. The limits of detection (LODs) obtained for Cu and Zn were 340 ng g–1 and 140 ng g–1, respectively, while LODs of 12 ng g–1 and 7 ng g–1 were determined for Pb and U, respectively. LA-ICP-MS was used to produce images of elements in 20 μm thin tissue sections of primary human brain tumours [glioblastoma multiforme (GBM)] and adjacent nonneoplastic brain tissue. The concentration and distribution of selected elements (Cu, Zn, Pb and U) are compared with the

control tissues and regions affected by GBM. A correlation was found between LA-ICP-MS and receptor–autoradiographic results. For receptor–autoradiographic techniques, labeling for A1AR and the pBR was employed. Using a large laser ablation chamber, commercially available from Cetac Technologies, we quantitatively analyzed the Zn and Cu distribution in a 20 µm thin section of the whole human hemisphere (110 mm × 65 mm) and found a layered structure of cortex with higher metal concentration in the grey matter compared to the white matter. In addition, the selenium distribution in a thin section of tissue was quantitatively measured by LA-ICP-MS. In our experiments, snails were fed with 1000 µg mL–1 selenium (Na2SeO3) solution for 60 h. For the quantification of selenium, matrix-matched standard reference materials were prepared and analyzed. The selenium distribution is illustrated, together with the Cu ion distribution, in part of the cross-section of a snail in Figure 4. A selenium concentration in a 100 µm thin section of snail tissue was observed to be up to 250 µg g–1. The detection limit for selenium was found to be 150 ng g–1. In order to improve the lateral resolution of LA-ICPMS to the nanometre scale range, near-field LA-ICP-MS



Imaging Mass Spectrometry in Medical and Biological Tissues with LA-ICP-MS

(a)

Ø (b)

Figure 2. (a) Transient signals of 31P+, 63Cu+, 56Fe+ and spectrum of protein spot 6 (creatine kinase, b chain).

Zn+ in protein spots 7–9 measured by LA-ICP-MS; (b) MALDI-FT-ICR mass

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(NF-LA-ICP-MS) was created in the authors’ laboratory.26 This technique uses the near-field enhancement effect at 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 the electrolyte. For nanolocal analysis by NF-LA-ICP-MS on soft matter (for example, on 2D gels and biological samples) a small-volume transparent laser ablation chamber was constructed and coupled

to a double-focusing sector field ICP mass spectrometer. A small amount of soft sample material was ablated at atmospheric pressure by a single laser shot in the near field of the silver tip in the defocused Nd:YAG laser beam. By singleshot analysis on 2D gels and biological surfaces doped with uranium, an enhancement of ion intensities of transient signals compared to a background signal of up to factor 60 was observed. Using the near-field effect in LA-ICP-MS, it was demonstrated that nanolocal analysis is possible in

Figure 3. Distribution of Zn and Cu in the insular region of the human brain measured by LA-ICP-MS.

J.S. Becker et al., Eur. J. Mass Spectrom. 13, 1–6 (2007) 

Figure 4. Distribution of Se and Cu in a cross-section of a part of a snail measured by LA-ICP-MS.

single-shot measurements of elements on biological samples and on a gel surface with nanometer-scale spatial resolution. In addition, 235U+ / 238U+ isotope ratio measurements on a gel doped with isotope standard reference material, NIST U020, were performed by NF-LA-ICP-MS. The 235U+ / 238U+isotope ratio (average of seven single shot measurements) was found to be 0.017 ± 0.002. These first experiments on near-field LA-ICP-MS will be of great importance, in order to produce, in future, the ­distribution profiles of the measured elements as well as to study the fine structures in the analyzed biological tissues. In present studies, basic investigations in the development of NF-LA-ICP-MS were performed and the technique will be applied to study essential and toxic element distributions within tissue cross-sections, tissue compartments, single cells, cell organelles and protein extracts. NF-LA-ICP-MS could open up a challenging path for future applications in nano-imaging of elements in life sciences, biology and medicine, for example, for analyses of single cells, cell organelles or biological structures in the nanometer range in order to detect disease, but also in materials science, nano­technologies and nanoelectronics.

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Imaging Mass Spectrometry in Medical and Biological Tissues with LA-ICP-MS

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Received: 28 November 2006 Revised: 26 January 2007 Accepted: 29 January 2007 Publication: 23 May 2007