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ICP-MS and Elemental Tags for the Life Sciences

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ICP-MS and Elemental Tags for the Life Sciences Dipl.-Chem. Charlotte Giesen ICP-MS and Elemental Tags for the Life Sciences BAM-Dissertationsreihe • Band 83 Berlin 2012 Die vorliegende Arbeit entstand an der BAM Bundesanstalt für Materialforschung und -prüfung. Impressum ICP-MS and Elemental Tags for the Life Sciences 2012 Herausgeber: BAM Bundesanstalt für Materialforschung und -prüfung Unter den Eichen 87 12205 Berlin Telefon: +49 30 8104-0 Telefax: +49 30 8112029 E-Mail: [email protected] Internet: www.bam.de Copyright © 2012 by BAM Bundesanstalt für Materialforschung und -prüfung Layout: BAM-Referat Z.8 ISSN 1613-4249 ISBN 978-3-9814634-7-7 ICP-MS and Elemental Tags for the Life Sciences D i s s e r t a t i o n zur Erlangung des akademischen Grades d o c t o r r e r u m n a t u r a l i u m (Dr. rer. nat.) im Fach Chemie eingereicht an der Mathematisch-Naturwissenschaftlichen Fakultät I der Humboldt-Universität zu Berlin von Dipl.-Chem. Charlotte Giesen, geb. Peter geb. am 18. Juni 1982 in Marburg Präsident der Humboldt-Universität zu Berlin Prof. Dr. Jan-Hendrik Olbertz Dekan der Mathematisch-Naturwissenschaftlichen Fakultät I Prof. Dr. Andreas Herrmann Gutachter/innen: 1. Prof. Ulrich Panne 2. Prof. Michael W. Linscheid 3. Prof. Detlef Günther Tag der mündlichen Prüfung: 20.12.2011 Zusammenfassung Zusammenfassung Die induktiv gekoppelte Plasma Massenspektrometrie (ICP-MS) wurde aufgrund ihrer hohen Empfindlichkeit, des großen linearen dynamischen Messbereichs und ihrer Multielementfähigkeit für die Analytik von Biomolekülen eingesetzt. Jedoch wird das Potential dieser Technik außerhalb der ICP-Gemeinschaft selten genutzt. Daher wurden in dieser Arbeit ICP-MS-basierte Immunoassays für medizinische (Krebsdiagnostik, Toxizitätsstudien zu Cisplatin), biochemische (DNA Mikroarray, Einzelzellanalytik) und umweltrelevante (Lebensmittelanalytik) Anwendungen entwickelt. Die Detektion erfolgte durch chemische Markierungen. Die Laserablation (LA)-ICP-MS wurde für die direkte Analyse von festen Proben wie Mikroarrays und Gewebedünnschnitten eingesetzt. Ein Immunoassay zur Ochratoxin A (OTA) Bestimmung in Wein wurde entwickelt, und die ICP-MS mit der herkömmlichen photometrischen Detektion verglichen. Die Nachweisgrenze betrug 0.003 µg L-1, und der Quantifizierungsbereich lag zwischen 0.01 und 1 µg L-1 für beide Methoden. Für die LA-ICP-MS basierte DNA Mikroarray Detektion wurden Goldnanopartikel über Streptavidin-Biotin Bindungen eingeführt. In der immunhistochemischen Diagnostik werden üblicherweise für einen Patienten bis zu 20 Krebsmarker abgefragt, was zu einer Reihe von zeitaufwändigen Färbeprotokollen führt. Daher wurde hier die LA-ICP-MS als eine neue, multiplexfähige Detektionsmethode für die Analytik an Gewebeschnitten entwickelt. Hierzu wurden Lanthanide für die Detektion von bis zu drei verschiedenen Tumormarkern in Brustkrebsgewebe eingesetzt. Darüber hinaus wurde mittels Iodmarkierung eine LA-ICP-MS Methode entwickelt, in der ein 4 µm Laserstrahl ausreichend war für die Darstellung von einzelnen Zellen und Zellkernen. Iod wurde außerdem als interner Standard für Gewebeschnitte verwendet. Zusätzlich wurden Pt-Protein Komplexe mit 1D und 2D Gelelektrophorese getrennt und mit LA-ICP-MS analysiert. Die hohe räumliche Auflösung dieser Technik wurde anhand der Detektion von platinierten Proteinen in Rattennierengewebe auch in einer aktuellen Studie zur Toxizität von Cisplatin und dem daher notwendigen Schutz der Niere unter Beweis gestellt. v Abstract Abstract Inductively coupled plasma mass spectrometry (ICP-MS) has been applied for the analysis of biomolecules due to its high sensitivity, wide linear dynamic range, and multielement capabilities. However, outside the elemental MS community the potential of this technique, e.g. for life sciences applications, is not yet fully exploited. Thus, the development of ICP-MS-based (immuno) assays for a wide range of medical (cancer diagnostics, cisplatin toxicity studies), biochemical (DNA microarray, single cell analysis), and environmental (analysis of comestible goods) applications was accomplished by utilization of chemical labels. Laser ablation (LA)-ICP-MS was employed for the direct analysis of solid samples like microarrays and thin tissue sections. An immunoassay was developed for ochratoxin A (OTA) determination in wine, and ICP-MS detection was compared to conventional photometry by gold nanoparticle tagging and horseradish peroxidase, respectively. Detection limits of the assay were optimized to 0.003 µg L-1, and the quantification range was 0.01–1 µg L-1 for both methods. For LA-ICP-MS-based DNA microarray detection, gold nanoparticle tags were specifically introduced via a streptavidin-biotin linkage. In immunohistochemistry (IHC), 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. Hence, LA-ICP-MS was elaborated as a detection tool for a novel, multiplexed IHC analysis of tissue sections. Different lanthanides were employed for the simultaneous detection of up to three tumor markers (Her 2, CK 7, and MUC 1) in a breast cancer tissue. Additionally, iodine was employed as a labeling reagent, and a new LA-ICP-MS method for single cell and cell nucleus imaging was developed at 4 µm laser spot size. Iodine was also applied as a new internal standard for tissue samples. Moreover, Pt-protein complexes separated by an optimized 1D and 2D gel electrophoresis were analyzed by LA-ICP-MS. The high spatial resolution of this technique was further demonstrated in a current study of cisplatin toxicity and renal protective strategies in rat kidney tissue by detecting platinated proteins vii Contents Contents Zusammenfassung v Abstract vii Contents ix List of Abbreviations xv Part A: Introduction 1 A.1 Objective 2 Part B: Fundamentals 5 B.1. State of the Art 5 B.1.1 Elemental Tagging of Biomolecules and Detection by ICP-MS 6 B.1.2 Biomolecule Detection by LA-ICP-MS 9 B.1.2.1 Analysis of Tissues by LA-ICP-MS 11 B.1.3 Histology and Immunohistochemistry 13 B.1.4 Cisplatin 15 B.2. Mass spectrometry techniques 16 B.2.1 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) 16 B.2.1.1 Sample Introduction 17 B.2.1.2 Plasma Formation 18 B.2.1.3 Interface 19 B.2.1.4 Mass Analyzer 20 B.2.1.5 Slit Widths and Resolution 23 B.2.2 Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) 24 B.3 DNA Microarrays 26 B.4 Immunochemistry 27 B.4.1 Immunoassays 29 B.4.2 Immunohistochemistry (IHC) 31 B.5 Gel electrophoresis 31 ix Contents Part C: Experimental 35 C.1 Immunoassay 36 C.1.1 Materials 37 C.1.2 Buffers and Chemicals 37 C.1.3 Antibodies 39 C.1.4 Wines 39 C.1.5 Immunoassay Procedure 39 C.1.6 ICP-MS Detection 40 C.2 DNA Microarrays 41 C.2.1 Chemicals and Materials 42 C.2.2 Sample Preparation 45 C.3 Immunohistochemistry 47 C.3.1 Labeling of Primary Antibodies 48 C.3.1.1 Chemicals 48 C.3.1.2 Purification of Antibodies 48 C.3.1.3 Production of Metal Chelate 49 C.3.1.4 Antibody Labeling 50 C.3.2 Immunohistochemical Reaction 50 C.3.2.1 Conventional IHC staining 50 C.3.2.2 IHC for LA-ICP-MS 52 C.3.2.3 LA-ICP-MS of Breast Cancer Tissue Sections 52 C.4 Labeling of Single Cells and Tissues by Iodination 55 C.4.1 Iodination of Thin Sections 55 C.4.2 Fixation and Iodination of Fibroblast Cells 55 C.4.3 LA-ICP-MS of Iodinated Cells and Tissue Sections 56 C.4.4 Thyroid Gland 58 C.5 Gel Electrophoresis 58 C.5.1 Chemicals 58 x BAM-Dissertationsreihe Contents C.5.2 Incubation of Standard Proteins with Cisplatin 58 C.5.3 Cell Cultures and Protein Extraction 59 C.5.4 One Dimensional Electrophoresis (SDS-PAGE) 59 C.5.5 Two Dimensional Electrophoresis (2-DE IEF + SDS-PAGE) 60 C.5.6 Protein Fixation and Staining 62 C.5.7 Gel Drying 62 C.5.8 LA-ICP-MS of Dried Gels 62 C.6 Study of Renal Protection in Rats Treated with Cisplatin 65 C.6.1 Drugs 65 C.6.2 Rat Kidney Sample Preparation 65 C.6.3 HE Staining 66 C.6.4 LA-ICP-MS of Rat Kidney Tissue 67 Part D: Results and Discussion 69 D.1 ICP-MS-linked Immunoassay for Ochratoxin A Determination in Wine 69 D.1.1 Minimization of Nonspecific Binding 71 D.1.2 Immunoassay Digestion for ICP-MS Detection 73 D.1.3 Optimization of Wine Sample Detection 74 D.1.4 Figures of Merit for Photometry and ICP-MS Detection 77 D.1.5 Summary 80 D.2 DNA Microarray Detection by LA-ICP-MS 81 D.2.1 Optimization of Microarray Preparation 82 D.2.2 Single Pulse LA-ICP-MS 85 D.2.3 LA-ICP-MS Analysis of DNA Microarrays 86 D.2.4 Summary 89 D.3 Combination of Immunohistochemistry with Detection by LA-ICP-MS 90 D.3.1 Labeling with SCN-DOTA: Optimization of Sample Preparation and LA-ICP-MS Measurements 91 D.3.1.1 Optimization of Tissue Thickness and Laser Energy 91 D.3.1.2 Optimization of Incubation Time and Antibody Concentration 93 xi Contents D.3.1.3 Optimization of LA-ICP-MS Parameters 95 D.3.1.4 Selectivity of Labeled Tumor Markers 98 D.3.2 Multiplex IHC 104 D.3.2.1 Multiplexed Detection of Her 2, CK 7, and MUC 1 in Breast Cancer Tissue 104 D.3.2.2 Comparison of MUC 1 with Cu and Zn Distribution in Breast Cancer Tissue 107 D.3.3 Summary 109 D.4 Iodine as an Elemental Label for Imaging of Single Cells and Tissue Sections by LA-ICP-MS 110 D.4.1 Iodination of Fibroblasts 112 D.4.2 Optimization of Tissue Labeling by Iodination 113 D.4.3 Iodination of Liver Biopsy Tissue 114 D.4.4 A New Internal Standard for Tissue Sections 116 D.4.5 Summary 119 D.5 LA-ICP-MS Detection of Platinum-bound Proteins separated by 1D- and 2D-Gel Electrophoresis 120 D.5.1 Optimization of 1D-SDS-PAGE for LA-ICP-MS 121 D.5.2 Identification of Platinated Protein Spots in
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