ICP-MS and Elemental Tags for the Life Sciences

Dipl.-Chem. Charlotte Giesen

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

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. .

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 2D-GE by LA-ICP-MS 124

D.5.3 Summary 126

D.6 Imaging of Metal Distribution in Rat Kidneys by LA-ICP-MS 127

D.6.1 Imaging of Platinum Distribution 127

D.6.1.1 Optimization 127

D.6.1.2 Evaluation 129

D.6.1.3 Estimation of Total Pt Amount in Kidney Tissue 132

D.6.2 Copper and Zinc Bioimaging 134

D.6.3 Platinum Bioimaging for the Evaluation of a Nephroprotector 135

D.6.4 Summary 137

E Summary and Outlook 139

xii BAM-Dissertationsreihe Contents

F Appendix 143

F.1 Nano Electrospray Ionization Time-of-Flight Mass Spectrometry (nESI-TOF-MS) 143

F.2 Analysis of MUC 1 (Tb) by nESI-Q-TOF-MS 144

F.3 Application of Highly Amplifiying Labels to LA-ICP-MS-based IHC 147

F.4 Gold Nanoparticle Incubation of Fibroblast Cells and Detection by LA-ICP-MS 151

F.5 Imaging of Thyroid Gland Tissue by LA-ICP-MS 152

Acknowledgments 155

Literature 159

xiii

Abbreviations

List of Abbreviations % Percent µ Micro (10-6)

1D One dimensional 2D Two dimensional Anti-CK 7 Monoclonal mouse anti-human cytokeratin 7 Anti-Her 2 Polyclonal rabbit anti-human c-erbB-2 oncoprotein Anti-MUC 1 Monoclonal mouse anti-human mucin 1 Anti-TTF 1 Monoclonal mouse anti-thyroid transcription factor 1 APS Ammonium persulfate Ar Argon Au Gold B Magnetic field Ba Barium BSA Bovine serum albumin bw Body weight

CA Carbonic anydrase from bovine erythrocytes CBB Coomassie-Brilliant Blue CK 7 Cytokeratin 7 CK 7 (Tm) Anti-CK 7 labeled with Tm loaded DOTA cm Centimeter

CO2 Carbon dioxide cps Counts per second Cu Copper CYT c Equine cytochrome c Da Dalton DMF N,N-Dimethylformamide

DNA Deoxyribonucleic acid DOTA 1,4,7,10-Tetraazacyclododecane-1,4,7,10-tetraacetic acid DTT Dithiothreitol E Electrostatic field

xv Abbreviations e.g. Exempli gratia, for example

ELISA Enzyme-linked immunosorbent assay

ESA Electrostatic analyzer

ESI Electrospray ionization et al. Et alii ETH Eidgenössisch Technische Hochschule eV Electron volt f Femto (10-15)

F Force

Fab Antibody fragment antigen binding site

Fc Antibody fragment crystallizable

FFPE Formalin-fixed, paraffin-embedded g Gravity of Earth GE Gel electrophoresis H Height h Hour

HCl Hydrochloric acid He Helium HE Hematoxylin eosin Her 2 (Ho) Anti-Her 2 labeled with Ho loaded DOTA Ho Holmium HSA Human serum albumin Hz Hertz I Iodine ICAT Isotope-coded affinity tags ICP Inductively coupled plasma

IEF Isoelectric focusing IgG Immunoglobulin G

IHC Immunohistochemistry IPG Immobilized pH gradient Ir Iridium xvi BAM-Dissertationsreihe Abbreviations iTRAQ Iobaric tags for relative and absolute quantification J Joule Ki Potassium iodide

KI3 Potassium triiodide L Length L Liter LA Laser ablation

LC Liquid chromatography

LOD Limit of detection

LSB Lämmli sample buffer m Milli (10-3)

M Molar (mol L-1) mA milli Ampere MALDI Matrix-assisted laser desorption / ionization MeCAT Metal-coded affinity tag Milli-Q Ultrapure water (> 18 MΩ) by Millipore min Minute mm Millimeter MS Mass spectrometer

MUC 1 (Tb) Anti-MUC 1 labeled with Tb loaded DOTA MUC 1 Mucin 1

MW Molecular weight

MYO Horse heart myoglobin n Nano (10-9)

Na2S2O4 Sodium dithionite NaCl Sodium chloride nm Nanometer

NWR New Wave Research O.D. Optical density

OTA Ochratoxin A OTB Ochratoxin B

xvii Abbreviations p Pico (10-12)

PBS Phosphate buffered saline

PDA Piperazine diacrylamide PEG Polyethylene glycol pH Negative logarithm (base 10) of the molar concentration of + dissolved hydronium ions (H3O ) pI Isoelectric point PMSF Phenylmethylsulfonyl fluoride PROTEIN-AQUA Protein absolute quantification p-SCN-Bn-DOTA Para-2-(4-Isothiocyanate-benzyl)-DOTA r Radius R Resolution RF Radio frequency rpm Revolutions per minute

RSD Relative standard deviation RT Room temperature s Second

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis SILAC Stable isotope labeling by amino acids in cell culture SSC Saline sodium citrate t Time Tb Terbium TBAA Tetrabutyl ammonium acetate TEMED Tetramethylethylenediamine TF Transferrin Tm Thulium TMB 3,3’,5,5’-Tetramethylbenzidine

TOF Time-of-flight

Tris Tris(hydroxymethyl)aminomethane

Tris-NO3 Tris-nitrate TTF 1 Thyroid transcription factor 1 v Velocity xviii BAM-Dissertationsreihe Abbreviations

V Volt v / v Volume fraction W Watt W Width Zn Zinc

xix

Part A: Introduction

Part A: Introduction The human genome is precisely defined by its 2.85 billion nucleotides,1 and the mapping of approximately 22 500 genes2 has enabled the tracing of diseases to their molecular causes. Insights into genetically determined differences in drug metabolism have generated new diagnostic and preventive options for personalized medicine.3 On the other hand, our 22 500 genes encode for approximately ten million proteins with a diversity of posttranslational modifications and varying concentrations (1-106 copies per cell).4 The protein expression patterns reflect current cell and environmental conditions and thus, the proteome (‘the protein complement of the genome’5) is highly dynamic. The process of studying the proteome is known as proteomics,5 and with the invention of ESI6 and MALDI7 for the ionization of peptides and proteins, mass spectrometry became the main workhorse in proteomics.

Immunoassays are employed for the in situ detection of proteins by means of a specific antibody.8 A secondary antibody, usually coupled to a chromogenic enzyme complex,9, 10 is applied for signal amplification. Such conjugates have also been used histochemically to detect antigens in tissue sections.11 The immunohistochemical (IHC) staining allows an assessment of the expression level, and the intracellular localization of a target protein. However, the techniques for biomolecule detection mainly rely on luminescence, photometry or colorimetry,4 and are rarely standardized.12 This is due to a number of factors: the matrix effects, the inherent difficulty of quantification owing to both a species- and environmental-specific response, and the lack of powerful multiplexing strategies for connecting different variables within a biological system.

The separation of proteins in a complex sample can be performed by 2D-gel electrophoresis (GE).13, 14 The resolving power of this technique is based on the graded mobility of charged molecules of different size in an electric field. After tryptic digestion of selected protein spots, the resulting peptides are usually separated by liquid chromatography (LC), and subsequently analyzed by molecule specific mass spectrometry. In addition to protein identification, quantitative proteomics is aimed at the determination of protein quantity or quantitative change in a complex sample.9, 10 This provides new insights into the functionality of a living organism, facilitates the

1 Part A: Introduction identification of disease markers, and contributes to the discovery of proteins as therapeutic targets.15 The combination of LC-MS/MS with artificial tags (labels) e.g., stable isotope labeling by amino acids in cell culture (SILAC),16 isotope-coded affinity tags (ICAT),17 or isobaric tags for relative and absolute quantification (iTRAQ),18 yields semi-quantitative data, and started the era of quantitative proteomics. Nevertheless, this approach still poses a challenge to methodologies for absolute quantification like PROTEIN-AQUA (protein absolute quantification),19 since standards need to be synthesized for any investigated peptide (upon tryptic digestion, a single protein generates 30–50 different peptides5).

On the contrary, inductively coupled plasma mass spectrometry (ICP-MS) has been applied more and more often for the (quantitative) analysis of biomolecules due to its high sensitivity, which is independent of structure,20 wide linear dynamic range, and multielement capabilities.21 When spatial information is required for a solid sample, laser ablation (LA)-ICP-MS is the method of choice for the direct detection of heteroelements. This technique is especially useful for the analysis of thin tissue sections to sustain morphological information.

A.1 Objective The detection of proteins by for example, sulphur (methionine or cysteine moieties), phosphorus (phosphoproteins), or metal heteroatoms, has become an established method during the last ten years.21 Apart from the detection of natural tags, any biomolecule of interest can be analyzed by use of elemental labels.21 The use of different metal labels for the detection of biomolecules enables the discrimination of various parameters within a single experiment and hence, the analysis of a complex system. However, only a few groups have reported on the multiplexed analysis of biomolecules via elemental labeling.21

Methods based on ICP-MS offer a promising quantification concept, low matrix effects compared to conventional bioanalytical techniques due to the independency of structure, and biologically relevant limits of detection (LODs) in the low pg g-1 range.22 Beyond that, the use of metal tags in combination with ICP-MS provides equivalent sensitivity to identically tagged biomolecules,23 which is a prerequisite for

2 BAM-Dissertationsreihe Part A: Introduction quantification. Therefore, it is an ideally suited tool to complement molecule specific MS-based methods in quantitative proteomics.

Outside the elemental MS community the potential of ICP-MS is not yet fully exploited, e.g., for proteomics and genomics applications.20, 24 It is therefore the aim of this work to transfer the advantages of this technique to current challenges in the life sciences, and to demonstrate their suitability to real samples.

One of the main advantages of ICP-MS, its multielement capability, can be analytically exploited for the development of multiplex assays, which enable the simultaneous detection of several analytes, or experimental parameters within a single experiment. Thus, the development of novel 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 is a main goal of this work. Different metal tagging approaches – implementing nanoparticles and complexated lanthanide ions – can be employed, for example, for the detection of toxins, tumor markers, metalloproteins, and metallodrugs. Insight into the distribution of these biomolecules can help to design more effective drugs with lower toxicity and side effects. Furthermore, the application of multiplex assays can speed up medical diagnostics. This is not only of high benefit to the individual patient, but is of great economic interest since it decreases the cost of health care.

Part B: Fundamentals

B.1. State of the Art Inductively coupled plasma mass spectrometry combines fast, highly sensitive, and multi element analyses with a wide linear dynamic range. These characteristics make it especially useful for the detection of proteins and other biomolecules, which are expressed with a diversity of modifications at varying concentrations (102–106 copies per cell).25 Metalloproteins, which play a key role in many biological processes, account for one third of the whole proteome,26 and can be detected by ICP-MS in a straightforward way. However, it features a structure-independent detection, and molecule specific MS is needed for protein identification. On the other hand, this limitation provides one of the main advantages of ICP-MS: the structure-independent detection offers low matrix effects in biological samples, and reduces the complexity of the sample. The benefits of combining molecular MS with elemental MS have been recently reviewed by Becker and Jakubowski.24

In the following, the term relative quantification is referred to the comparison of signal intensities, and the term absolute quantification is referred to quantitative data, which allow the conversion of signal intensity into a corresponding concentration level.21 In recent years, ICP-MS has been applied more and more often for the relative and absolute quantification of biomolecules, and an overview on the use of heteroatoms and chemical labels for the quantitative MS analysis of biomolecules is given by Prange and Pröfrock.21 The detection of proteins by elements like sulphur (methionine or cysteine moieties) and phosphorus (phosphoproteins, DNA), often coupled to HPLC, has become an established method during the last ten years. Moreover, the derivatization with artificial, elemental tags (labels) enables the detection of virtually any biomolecule of interest, and the discrimination of various parameters within a single experiment.

The low elemental detection limits in the pg g-1 range, the possibility for absolute quantification, and the multi element capabilities make ICP-MS an ideal detection method for bioanalytical techniques like immunoassays, facilitating a high throughput format. Furthermore, the application of LA-ICP-MS is advantageous for the analysis of solid samples, since it provides high lateral resolution in the low µm range,27

5 Part B: Fundamentals combined with the virtual lack of sample preparation. In the life sciences, this is of high benefit for the analysis of tissue samples, and the screening for a variety of different antigens and metalloproteins.28

B.1.1 Elemental Tagging of Biomolecules and Detection by ICP-MS The first element-tagged immunoassay was described in 2001 by Zhang et al. for the determination of thyroid hormones in human serum by means of a sandwich-type immunoreaction.29 The authors employed biotinylated antibodies, and Eu3+-labeled streptavidin. In 2002, the same group described the determination of thyroxin in a competitive immunoassay.30 The concentration of thyroxin was determined by its ability to inhibit the binding of Eu-labeled thyroxin to the corresponding monoclonal antibody. Owing to the enrichment of antigen on a microwell surface, a detection limit of 7.4 ng mL-1 was achieved in 25 µL sample, with a working range up until 233 ng mL-1. This result is comparable to an enzyme immunoassay for thyroxin with photometric detection, which provides a working range of 10–240 ng mL-1.31 Apart from lanthanides, gold nanoparticle tags were applied for labeling. Baranov et al. implemented a gel-filtration-based ICP-MS immunoassay to separate and quantify different labeled antibodies;32 Zhang et al. employed antibodies conjugated with colloidal gold nanoparticles for a sandwich-type immunoreaction.33 In a study of nanoparticle atomization by ICP-MS, the authors showed that the ICP-MS signal is not influenced by the organic matrix.33

The multiplexed determination of proteins using element-tagged immunoassays coupled with ICP-MS detection was pioneered by Tanner and co-workers.34 They implemented the Wallac AutoDELFIA™ reagents, which were originally designed for the time-resolved fluorescence detection of enzyme-linked immunosorbent assays (ELISAs). The limit of detection achieved by these reagents was 0.1–0.5 ng mL-1 of target proteins, which was about one order of magnitude inferior to the AutoDELFIA™ method.23 In the first multiplex assay, one antibody was labeled with Eu (Wallac AutoDELFIA™), and the second antibody was labeled with 1.4 nm nanogold clusters (NANOGOLD®).34 In 2006, Ornatsky et al. presented a 4-plex assay in a human leukaemia cell line model by using Sm, Eu, and Tb labels (Wallac AutoDELFIA™) in combination with a NANOGOLD® reagent.35 The same procedure

6 BAM-Dissertationsreihe Part B: Fundamentals was also applied for mRNA detection.36 An ICP-MS-based immunoassay using Eu-tagged antibodies was designed by Careri et al. for the detection of hidden peanut allergens in foods.37 The limit of detection calculated for raw peanut protein extracts was 1.5 ng mL-1. In a cereal matrix, the limit of detection for peanuts was approximately 2 mg kg-1. The authors illustrated that the LOD is influenced by antibody binding efficiency and nonspecific binding, since the ICP-MS instrumental LOD for Eu-labeled antibody was 0.1 ng mL-1.37

Another possibility for introduction of the metal tag is the ligand DOTA (1,4,7,10- tetraazacyclododecane-tetraacetic acid), which was applied by Whetstone et al. for the differential labeling of peptides.38 The authors showed that this approach was suitable for reversed phase chromatography separation and subsequent MS/MS experiments. The stability constants (K) of DOTA complexes and lanthanide fluorides are given in Table B.1.1-1.39 The high log K (DOTA) values for lanthanides result in particularly stable complexes. Hence, as has been verified by Waentig et al., there is no detectable exchange of lanthanides between the different complexes.40 This provides a variety of choices for metal tags to be employed in multiplex experiments. A clinical application in a multiplexed ICP-MS determination of cancer biomarkers in serum and tissue lysates was investigated by Terenghi et al.41 They used size exclusion chromatography (SEC) to separate the antibody-antigen complex from unbound species. With this method, a discrimination of ovary and uterus tumor tissue and control samples was achieved.

7 Part B: Fundamentals

Table B.1.1-1: Stability constants of lanthanide DOTA complexes (log K (DOTA)) and lanthanide fluorides (log K (Fluoride)).39

Lanthanide log K (DOTA) log K (Fluoride)

La 22.86 2.67

Ce 23.39 2.81

Pr 23.01 3.01

Nd 22.99 3.09

Sm 23.04 3.12

Eu 23.45 3.19

Gd 24.67 3.31

Tb 24.22 3.42

Dy 24.79 3.46

Ho 24.54 3.52

Er 24.43 3.54

Tm 24.41 3.56

Yb 25 3.58

Lu 25.41 3.61

In 2005, Krause et al. applied for a patent for a DOTA-based reagent with a cysteine reactive maleimide group, and a biotin modification for purification and enrichment of labeled peptides via biotin-avidin affinity chromatography, named metal-coded affinity tag (MeCAT).42 In a study by Ahrends et al. reaction parameters were optimized, and MeCAT was used to analyze proteins of the Sus scrofa eye lens in a model system.43 It was the first time that lanthanide labeling approaches for ICP-MS detection were combined with a proteomic workflow for protein identification. The authors proved that the same differentially labeled peptides co-elute during LC-ESI-MS/MS, which is a prerequisite for multiplexed proteomics. Hence, the low matrix effects of biological samples in ICP-MS, and the use of simple, non-species specific metal standards opened up new possibilities for absolute quantification of proteins and peptides, which can be identified by molecule specific MS in a parallel experiment.

8 BAM-Dissertationsreihe Part B: Fundamentals

Furthermore, an investigation of variations in an Escherichia coli proteome due to different growth temperatures was conducted with this technique.44 The first implementation of MeCAT to absolute protein quantification was published recently.45

For signal amplification in ICP-MS, Tanner and co-workers developed a maleimide-functionalized polymer tag, which is especially useful for the analysis of low abundant proteins.46 A landmark publication was the presentation of a new mass cytometer: Bandura et al. coupled flow cytometry to a high spectrum generation frequency ICP-TOF-MS, which is commercially available as the CyTOF (DVS Sciences Inc., Richmond Hill, Ontario, Canada).25 Such an instrument was employed for the simultaneous detection of 20 antigens on the surface of single cells of leukemia cell lines and leukemia patient samples, which is hard to achieve by quadrupole-based ICP-MS instruments. The differential labeling was accomplished by polymer tags, which were complexated with a variety of lanthanide isotopes. Recently, Bendall et al. reported on the simultaneous detection of a multiplex assay with 34 cellular parameters in the primary human hematopoietic system.47

Since the ICP signal is proportional to the number of labels attached, a further signal enhancement might be possible by use of lanthanide-coded particles, which in the past could not be applied for labeling in a straightforward way due to difficulties in biofunctionalization. Winnik et al. recently proposed a solution by growing a functional polymer shell onto the particle surface, which subsequently was biofunctionalized with NeutrAvidin.48

B.1.2 Biomolecule Detection by LA-ICP-MS For a variety of biochemical or medical applications it is important to sustain spatial information from a sample and thus, LA-ICP-MS-based methods are employed. The detection and quantification of phosphorylated proteins after gel electrophoresis and electroblotting by LA-ICP-MS was first described by Marshall et al.49 and Wind et al.50 In 2003, LA-ICP-MS was implemented by Chéry et al. for the detection of selenoproteins in red blood cell extracts and in selenized yeast after 1D and 2D sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), respectively.51 Further details on gel electrophoretic separation of metalloproteins, and the use of LA-ICP-MS as a detection tool are given in a review by Ma et al.52 The

9 Part B: Fundamentals use of LA-ICP-MS as a detection tool for SDS-PAGE of lanthanide labeled antibodies provides a LOD of about 65 pg,53 whereas a LOD of approximately 1 ng is achieved by gel silver staining.54 However, the application of SDS-PAGE with LA-ICP-MS detection is still hampered due to the high risk of metal losses during the sample preparation and separation procedure.55 Direct methods were also applied for immunoassays: For analysis of the Mre11 protein in crude lysates of CHO-K1 fibroblasts, Müller et al. employed LA-ICP-MS of Western Blot membranes using gold-cluster-labeled antibodies.56 They achieved a LOD of 0.20 amol due to the metal cluster, which enhanced sensitivity in correlation to the number of cluster atoms.56 Moreover, the multi element capability of LA-ICP-MS directly on a Western Blot membrane has been investigated by Waentig et al. using lanthanide labeled antibodies with LODs in the sub pmol range.57 Hu et al. applied antibodies conjugated with different lanthanide ions or nanoparticles, and reported on the detection of three proteins on a single microarray spot by means of LA-ICP-MS.58 The LODs were 0.20, 0.14, and 0.012 ng mL-1 employing Sm-labeled, Eu-labeled, and Au nanoparticle-labeled antibodies, respectively. The latter LOD reflects the nanoparticle label, which provides higher sensitivity in ICP-MS than Sm3+ or Eu3+ labels. The labeling of proteins with iodine is covalent, and thus is applicable to electrophoresis and electro-blotting.59, 60 Radioactive iodine has been first used by Hunter and Greenwood61 for labeling of hormones. Up to now, many other complex iodination agents have been applied,62-65 but the use of a saturated iodine solution in potassium iodide is less laborious and inexpensive. Direct labeling of proteins with iodine occurs at the two ortho-positions of tyrosine and at histidine residues. These electrophilic aromatic substitutions require reactive iodine species like triiodide, which is formed in aqueous solution from iodide and elemental iodine. The iodination of proteins and whole proteomes for analysis by LA-ICP-MS of Western Blot membranes was examined by Waentig et al.60

The investigations of metalloproteins, and the application of antibody tagging by nanoparticles or lanthanide containing chelates are also key topics in this work and will be described later in more detail.

10 BAM-Dissertationsreihe Part B: Fundamentals

B.1.2.1 Analysis of Tissues by LA-ICP-MS The bioimaging of tissue sections is increasingly applied in modern medicine as it can be an important tool for the interpretation of diseases, especially in cancer diagnostics.66 For this reason, bioimaging analytical techniques have been developed in the last years to monitor elemental and molecular distributions in tissues, as has been recently reviewed.28 Besides LA-ICP-MS,28, 67 these include scanning electron microscopy with energy-dispersive X-ray analysis (SEM-EDX),68 synchrotron X-ray fluorescence (SXRF),28 proton-induced X-ray emission (PIXE),69 secondary ion mass spectrometry (SIMS),28 and matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS).70 Metals are usually heterogeneously distributed in tissues or cells and thus, LA-ICP-MS seems to be the most convenient alternative for elemental bioimaging in tissue sections, since it provides multielemental detection with high sensitivity and high spatial resolution.67 Indeed, it has been widely used over the last decade for bioimaging of tissue samples.71,67

ICP-MS excels by its exceptional accuracy and ease of calibration, as has been mentioned before. However, elemental quantification of LA-ICP-MS is not straightforward and will be discussed in more detail in section B.2.2. Generally, calibration methods require the use of an internal standard to account for matrix dependence of the ablation process, variations in mass ablated, differences in transport efficiency, and instrumental drift.72 The isotope 13C is typically used as an internal standard in elemental bioimaging applications, despite its difference in mass and first ionization potential to many analytes, the possibility of abundance sensitivity effects, and a poor signal to noise ratio.73 An increase in 13C intensities is observed, which is linearly dependent on the mass ablated.73 However, it may be heterogeneously distributed in tissue samples owing to differences in the water content.74 Thus, the application of an alternative internal standard to correct for tissue inhomogeneities might be advantageous for thin tissue analysis.

Only very few reference materials are available for biological tissues, which hampers quantitative tissue bioimaging. Jackson et al. used pressed pellets of TORT-2 (lobster hepatopancreas), DOLT-2 (dogfish liver), and DORM-2 (dogfish muscle) for quantification of Cu, Zn, and Fe in rodent brains.75 For most other cases, laboratory standards are produced to permit matrix matching of standards to samples.

11 Part B: Fundamentals

Some authors have proposed quantification methods based on matrix-matched laboratory standards. Examples are spiked, frozen and embedded blood,76 spiked tissue homogenate,77 or solution-based calibration.77 With these methods, detection limits in the low µg kg-1 range were achieved for Th and U in human brain samples,77 and for Pt in rat brain.76 Furthermore, quantification using spiked thin polymer films was reported.78

The detection of non-metals such as sulfur and phosphorus,79 and the distribution of metals such as Cu, Zn, Fe, Li, K, and Na in tissue sections80 has been subject of many research projects. It was shown in the literature that tumor boundaries were clearly marked by imaging of 31P in lymph node biopsies.79 The mapping of metallodrugs by LA-ICP-MS is also possible.76, 81 Becker et al. used matrix-matched laboratory standards to receive information on the quantitative distribution of Cu, Zn, U, and Th in 20 µm thin tissue sections of human brain (hippocampus).77 Moreover, images of element distribution of metals (Zn, Cu, Fe, Mn, and Ti) in mouse heart tissue were compared to SIMS images of alkali metals and biomolecules. In a recent study by Wu et al., laser microdissection ICP-MS was used for imaging of heteroelements in brain tissue at laser spot sizes from 30-4 µm.82

The direct detection of cancer biomarkers in tissue sections is facilitated by labeling of antibodies, as has been discussed before. Hutchinson et al. applied Eu- and Ni-coupled secondary antibodies for LA-ICP-MS imaging of beta-amyloid deposits in mouse brain tissue.83 Furthermore, the distribution of mucin 1 (MUC 1) or human epidermal growth factor 2 (Her 2) in breast cancer tissue sections was studied by employing gold nanoparticle labeled secondary antibodies and silver enhancement for signal amplification.84 However, spatial resolution has been inferior to light microscopy due to a loss of sensitivity when using small laser spot sizes. Therefore, important information on tissue morphology is lost, as compared to hematoxylin and eosin (HE) staining in conventional histology. Obviously, the spatial resolution of tissue imaging in LA-ICP-MS has to be improved, and the number of simultaneously detected antigens has to be increased.

12 BAM-Dissertationsreihe Part B: Fundamentals

B.1.3 Histology and Immunohistochemistry For tumor diagnostics it is important to investigate morphology and composition of tissues or even further, of cells and cell compartments.85 However, tissue components are often difficult to distinguish by light microscopy due to their similar optical densities, but they can be visualized by the selective adsorption of dyes. Therefore, HE staining is widely used in histology. Hematoxylin imparts a blue color to cell nuclei, whereas eosin stains most other components pink. Additionally, immunohistochemistry (IHC) is employed in cancer diagnostics to assess the expression level and the intracellular localization of a tumor marker in tissue sections.

One third of all cancers in women are breast and ovary carcinoma. Together, they account for approximately ¼ of cancer related deaths in females.86 Three important tumor markers for breast cancer diagnosis, which are of interest in this work, are discussed in the following. The c-erbB-2 oncoprotein is overexpressed in 25 to 30 percent of human primary breast cancers.86 Anti-human epidermal growth factor receptor 2 (Her 2) recognizes an epitope on the c-erbB-2 protein, and is used as a primary antibody in IHC. The results of IHC analysis determine further medical treatment concepts. Another antibody used for IHC in breast cancer diagnosis is anti-cytokeratin 7 (CK 7). Cytokeratin 7 is a type II cytokeratin, which is specifically expressed in glandular epithelia. The majority of breast cancers are positive for CK 7 and it is used to determine the origin of metastatic breast carcinoma.87 Mucin 1, a membrane-associated protein found on the luminal surface of many columnar epithelia, is also known to be up-regulated in a subset of breast cancers, but is expressed at very low levels in normal mammary gland. Thus, anti-mucin 1 (MUC 1) is used as a tumor marker for breast cancer diagnosis.88

A drawback of the routine method relying on organic dyes is the lack of standardization, which in part is due to the inherent difficulty of quantification and reproducibility.89, 90 Although abundant data are available, results on the same gene product are often inconsistent. The frequency of Her 2 amplification and overexpression varied extensively for the same type of carcinoma (see Table B.1.3-1) owing to methodological and assessment variations between studies.89 Furthermore, the knowledge on tissue thickness is crucial for all histological investigations to guarantee reliable and reproducible results.91 In this context the

13 Part B: Fundamentals application of ICP-MS as a detection tool for IHC looks promising if an adequate internal standard can be applied, which is another challenge in this investigation.

Table B.1.3-1: Frequency of Her 2 expression (Determined by IHC or reverse transcriptase- polymerase chain reaction and Her 2 amplification in different tumor types as determined by fluorescent in situ hybridization or Southern hybridization. NSCLC: non-small-cell lung cancer.89 Tumor type Expression [%]

Breast carcinoma 13–100

Ovarian carcinoma 9–89

Colon carcinoma 0–83

Endometrial carcinoma 17–88

Gastric carcinoma 8–56

Head and neck cancers 0–93

NSCLC 4–100

Prostate carcinoma 0–100

Urinary bladder carcinoma 2–74

Commonly an indirect method is applied for IHC, using a primary antigen-specific antibody and a second incubation with an enzyme-labeled secondary antibody for signal amplification.92 This procedure impedes screening for simultaneous evaluation of more than one antigen at the same time, resulting in several subsequent staining procedures until a tumor-identifying antigen-profile can be determined. It is important to minimize specimen for IHC to maximize tissue for molecular studies.93 Hence, in modern cancer diagnostics it is urgently needed to screen for several tumor markers simultaneously, but the use of labeled secondary antibodies hampers a multiplexed IHC. There are only very few attempts of double or triple staining in IHC literature due to the risk of cross reactions among individual staining steps and the difficulty of simultaneous visual evaluation of stained tissue. A commercial triple staining cocktail (PIN-4 cocktail, BioCare) was employed for prostate carcinoma diagnosis. Each marker stained different cell compartments, but visualization of all three markers was

14 BAM-Dissertationsreihe Part B: Fundamentals accomplished by 3,3’ diaminobenzidine (DAB) in brown. Therefore, an independent assessment of three tumor markers was impeded. For the use of multiple dyes, secondary antibodies with different selectivity have to be employed.94 This approach hampers multiplexing by the limited availability of different antibody types applicable to IHC. Furthermore, dyes overlap for co-localized tumor markers,94 and there is a danger of cross-reactivity. Hence, time consuming subsequent staining procedures of parallel thin sections are still the gold standard in IHC. Signal saturation in the tissue is rapidly achieved, causing a limited dynamic range for detection by means of organic dyes. Quantification is hampered due to difficulties in standardization.12

B.1.4 Cisplatin

Cisplatin (cis-[Pt(NH3)2Cl2]) is employed in more than half of the oncologic treatments,95 especially in solid tumors.96 Its antitumor properties97 are based on the binding and distortion of the DNA in the nucleus, resulting in tumor cell death by apoptosis or necrosis.98 Owing to the high reactivity of elemental platinum other biomolecules containing nucleophilic groups, such as proteins, react with the drug as well and play a key role in the transport, cell uptake, and excretion of the drug. 99 Kidney is the main route for cisplatin excretion, where it accumulates to a greater extent than in other organs.100 Consequently, severe side-effects of cisplatin like nephrotoxicity occur in one third of the patients,101 and are dose-limiting for these treatments.102 The accumulation of platinum mainly affects the renal tubules, which are affected by aptoptosis and necrosis.101 Therefore, a variety of nephroprotective approaches have been tested both in vivo and in vitro,101, 103 albeit protective effects are usually incomplete and the cytotoxic effect on tumor cells is not fully understood. However, information on Pt-bound proteins and metal distributions in the affected organs might help to understand the toxicity related to Pt accumulation.

There have been reports in the literature on the detection of metals by LA-ICP-MS in mouse kidney after cisplatin treatment.81 Moreover, another LA-ICP-MS methodology was applied for the quantification of Pt in rat brain tissue after carboplatin administration.76 However, in both cases, the treated animals were killed only a few hours after the administration of cisplatin, which is not enough time for significant Pt accumulation in tissues and renal damage.103 Hence, the LA-ICP-MS-based bioimaging of kidney tissues from rats treated with pharmacologic

15 Part B: Fundamentals doses of cisplatin is still of interest. The developed methodology should provide a spatial resolution sufficient to study metal accumulation within the renal substructures and its connection with renal damage.

B.2. Mass spectrometry techniques

B.2.1 Inductively Coupled Plasma Mass Spectrometry (ICP-MS) Inductively coupled plasma-mass spectrometry (ICP-MS) is nowadays the most widely used elemental technique for (ultra)trace determination104 and covers a broad range of environmental,105 geological,106 industrial,107 and bioanalytical applications.108, 109 The technique was pioneered by Houk and Gray in the 1980s.110 Its advantages stem from the ability to ionize virtually all elements in the periodic table in the ICP, a sample introduction into the plasma at ambient pressure, a wide linear dynamic working range of 8 to 9 orders of magnitude,111 low detection limits of approximately 0.1–1 pg g-1,22 and multielement capabilities.

An overview on the basic components of an ICP-MS, which are discussed in more detail in the following chapters, is given in Fig. B.2.1-1. The sample, usually in liquid form, is pumped into a nebulizer, where it is converted into a fine aerosol. Laser ablation (LA) is applied for solid sample introduction, where the sample is converted into an aerosol by the interaction with a laser beam. The aerosol is subsequently transported into the ICP torch, where the plasma is maintained by interaction of an electromagnetic RF field with a flow of argon gas. The sample is ionized in the plasma, and directed into the mass spectrometer via through an interface, and the ion optics. The focused ion beam then enters the mass analyzer for mass-to-charge separation, before it reaches the detector. It depends on the design of the mass spectrometer, if the ions are simultaneously sampled (Time-of-flight, TOF, instruments) or detected (multi collector instruments), or if their mass-to-charge ratio (m / z) is scanned in a sequential manner (quadrupole instruments, single collector instruments).

16 BAM-Dissertationsreihe Part B: Fundamentals

Detector MS Interface

ICP Torch

Mass Analyzer Ion optics Sample Aerosol

Copper Coil

RF-Generator Turbo Turbo Pump Pump

Mechanical Pump

Figure B.2.1-1. Scheme of ICP-MS, including the plasma formation within the ICP torch, the MS interface, the mass spectrometer, and the detector. Adapted from reference 22.

B.2.1.1 Sample Introduction ICP-MS is mainly combined with aqueous sample introduction, but the analysis of solid samples is also possible, the most prominent technique being LA-ICP-MS. For efficient atomization and ionization of the aqueous sample in the plasma, it is converted to an aerosol by a nebulizer, and larger droplets (d < 10 µm) are removed in a spray chamber. The fine aerosol represents only 1–2 % of the sample.22 More details are discussed in a review on pneumatic nebulizers and spray chambers.112, 113 Most importantly, the composition of the ion population in the plasma is proportional to the concentration of the analyte species in the original sample solution. In the case of LA, the solid sample is converted to a fine aerosol by the use of He as a carrier gas in the ablation chamber (for more details refer to section B.2.2). Subsequently, the aerosol is transported to the plasma by an Ar gas flow, where ionization occurs. The process of ion formation is displayed in Fig. B.2.1.1-1.

17 Part B: Fundamentals

Nebulization Desolvation Vaporization Atomization Ionization Detection

Liquid + sample Aerosol

Particles Molecules Atoms Ions Mass Spectrum

Solid sample

Nebulizer Spray Plasma MS (liquid) Chamber

Laser (solid)

Figure B.2.1.1-1. Scheme of processes starting from sample introduction, ion formation, to analysis. Adapted from Agilent Technologies.114

B.2.1.2 Plasma Formation The plasma, an electrically neutral gas made up of ions and free electrons, is formed by a gas flow in a torch, which consists of three concentric quartz tubes. Mostly argon is used to sustain the ICP because of its high ionization potential and its availability in high purity. Inert gases also have the advantage of minimal chemical reactivity with various analyte species. Argon is introduced at approximately 15 L min- 1 into the space between the outer and the center tubes (i) to sustain the plasma, and (ii) to prevent the quartz torch from melting. The sample is injected as an aerosol into the plasma through the injector. The space between the injector and the intermediate tube is used for the introduction of an auxiliary flow of argon gas, typically 1 L min-1, to assist in the formation of the plasma and to ensure that the plasma is forced away from the tip of the injector, preventing it from melting. The magnitude of each of the various flow rates affects the stability of the plasma and has to be optimized daily.

The torch is encircled at the top by the load coil, which is connected to a radiofrequency (RF) generator. The magnetic field generated by the RF power,

18 BAM-Dissertationsreihe Part B: Fundamentals typically 700–1500 W, through the load coil induces a current in the Ar gas stream after ignition by a spark of a Tesla coil, which generates a few ‘seed’ electrons. The plasma is sustained by a process known as inductive coupling. As these seed electrons are accelerated by the electromagnetic RF field, collisions with neutral gas atoms produce additional electrons and create the ionized medium of the plasma. The largest current flow occurs on the periphery of the plasma, which gives the ICP a distinctive annular shape. This doughnut-type structure ensures the efficient introduction of sample aerosol into the central channel of the plasma where temperatures between 5000 and 7000 K have been measured. Temperatures in the outer region of the plasma can be as high as 10 000 K. The formation of the plasma by ionization is approximated by the Saha-Eggert equation at local thermal equilibrium as a function of temperature and density.

The sample aerosol in the plasma undergoes desolvation, vaporization, atomization, excitation, and ionization. This process is displayed in Fig. B.2.1.1-1. Because the average ionization energy of the argon plasma is dominated by the first ionization potential of argon (15.76 eV), and most elements have a first ionization potential below 16 eV, the plasma will efficiently produce singly charged ions for virtually all elements. In addition, few doubly charged ions will be produced because most elements have second ionization potentials larger than that of argon. Notable exceptions are barium and strontium. Their second ionization potential lies below 16 eV so that Ba and Sr will have an appreciable probability of forming doubly charged ions.

B.2.1.3 Interface Ions that are produced by the ICP are representatively sampled and extracted from the plasma through a cooled interface, thereupon separated and measured by a mass spectrometer. Since the ICP is operated at atmospheric pressure, and the ions have to be analyzed in a high vacuum mass spectrometer, an interface between both components is necessary. The function of the interface is to sample ions produced in the ICP representatively at atmospheric pressure, and to facilitate their transport into the mass spectrometer at high vacuum. For instance, a sector field mass analyzer requires a vacuum of less than 10-7 mbar to minimize collision effects of the ions with the background atmosphere in the system. Ion sampling of the plasma is achieved by

19 Part B: Fundamentals the use of two metal cones. The outside sample cone is used to sample the atmospheric pressure plasma; the diameter of its orifice is approximately 1 mm. Ions produced in the plasma pass through this orifice and form an ion beam. The extracted plasma is evacuated to a pressure of 3 mbar22 in the interface by a vacuum pump, and undergoes a supersonic expansion owing to the sudden drop in particle density. By this, the extracted plasma is ‘frozen’ since no collisions occur and thus, the probability of gas phase reactions is low. The central beam passes through the skimmer cone, which is positioned in a distance between 10–15 mm behind the sampler cone in the MS interface. The skimmer cone has a smaller orifice of ~ 0.5– 0.8 mm in diameter.

Behind the skimmer, ion lenses are used to assist in the transport of positively charged ions as they leave the skimmer cone and are evacuated to approximately 10-3 mbar. By varying the potentials on each of these lenses, the ion beam can be collimated and focused. Because all ions have approximately the same velocity

(established by the Ar gas), their kinetic energy (Ekin) is essentially dependent on their mass:

Ekin = ½ mv², (1) where m is the ion mass and v is the velocity. Thus, the heavier ions will have higher energy than the light ions. This results in different optimal ion lens voltage settings for each element, and a compromise setting is usually selected for multielement analysis.

B.2.1.4 Mass Analyzer Ions produced in the ICP are separated by the use of a mass spectrometer according to their m / z. After separation, the individual ions are sequentially or simultaneously (multicollector spectrometer) directed to a detector, which measures their individual ion currents. Therefore, the detection of the m / z of the ion allows identification of the isotope or molecule being measured, whereas the magnitude of the ion current provides quantitation of the analyte in the original sample, assuming stoichiometric sampling.

20 BAM-Dissertationsreihe Part B: Fundamentals

The most common instruments in ICP-MS are equipped with a quadrupole mass analyzer (Q-ICP-MS) due to their fast scanning abilities and their low costs. These mass analyzers consist of four cylindrical rods arranged parallel to each other in a symmetrical configuration. The quadrupole is scanned by ramping the alternating (ac) and direct current (dc) voltages applied to the rods. All ions, except those with a specific m / z ratio, will be forced on a path where they collide with the quadrupole rods. Only those ions with a specific m / z will have a stable path through the rods and will exit at the other end towards the detector. Scan speeds using quadrupole mass filters can be high, i.e. full mass range scan in 0.1 s,111 because the alternating electrical field can be rapidly changed. With TOF-ICP-MS, even 30 000 full mass spectra can be recorded every second. The challenge here is a fast data processing. However, its sensitivity is inferior to that of a Q-ICP-MS by 1–2 orders of magnitude owing to its duty cycle.111

On the other hand, a sector field mass spectrometer offers higher sensitivity at unit mass resolution, and lower instrument background (< 0.2 cps)111 which results in lower LODs. Especially for biological samples, where the analyte of interest is often of low abundance, this feature is of great advantage. Furthermore, these instruments are capable of high resolution measurements. Thus the ICP-MS used throughout most of this work was a double focusing sector field instrument with reverse Nier- Johnson geometry (ElementXR, Thermo Fisher Scientific, Bremen, Germany), most often operated in a low mass resolution mode. The term ‘double focusing’ refers to the combination of a magnetic with an electrostatic mass analyzer; a reverse Nier- Johnson geometry describes the arrangement of the magnetic mass analyzer in front of the electrostatic mass analyzer.

A magnetic sector mass analyzer consists of a magnetic field, which establishes the separation of ions according to their m / z. A singly charged ion of mass m at velocity v, passing through a curved flight tube in a magnetic field (B) will experience a Lorentz force FL perpendicular to both the direction of trajectory and the magnetic field.

This is given by:

FL = Bzv. (2)

21 Part B: Fundamentals

Under the influence of this force, the ion is deflected from its initial straight flight path into a circular trajectory with a radius rm. The orbiting ion experiences a centrifugal force FC given by:

FC = mv² / rm. (3)

At equilibrium we have

v = Bzrm / m. (4)

To induce the ion to travel from the source into the magnetic field, it is usually accelerated through a potential difference V, which gives it a kinetic energy

½ mv² = Vz. (5)

The combination of these two equations describes the ion motion in a magnetic field:

m / z = r²mB² / 2V. (6)

Ions with different masses follow curved flight paths with different radii (rm), and there are two options to scan the mass range: (i) scan B, and holding V and rm constant, (ii) scan V, and holding B and rm constant. The latter is usually preferred because scanning the voltage is much faster than scanning the magnetic field, which suffers from hysteresis. A third option, realized in a multicollector, is to keep B and V constant, and to detect ions of different m / z at different values of rm with a position- sensitive detector.

Considering equation (4) in the following form:

mv / z = Brm, (7) it can be seen that the magnetic analyzer is in fact not mass selective but separates charged particles according to their momentum-to-charge ratio. Ions of identical mass do not necessarily have the same energy. Consequently, ions with different masses, having the same momentum, will be selected together. Because the ICP ion source produces ions of constant medium velocities with a wide distribution, a wide energy spread is observed for the ions.

The energy spread of the ion beam limits the resolving power of a magnetic sector analyzer. To overcome this problem in high-resolution mass spectrometry, energy

22 BAM-Dissertationsreihe Part B: Fundamentals focusing is required. An electrostatic analyzer is used, which consists of two curved plates with a voltage (V) applied between them. When ions pass through an electrostatic field (E), they travel in a circular path of radius re such that the electrostatic force balances the centrifugal force:

mv² / re = Ez. (8)

The radius re is dependent on the ion's kinetic energy. If the voltage (V) is varied, ions with corresponding kinetic energy will sweep across the exit slit of the energy analyzer.

By combining a magnetic-sector mass analyzer with an electrostatic analyzer (double-focusing mass spectrometer), a significant improvement in resolution is accomplished. The magnetic sector determines trajectories by both mass and energy (i.e., momentum). In contrast, the electrostatic analyzer establishes ion trajectories by energy only. The reverse Nier-Johnson geometry is desirable because it minimizes background noise. An ion traveling through the final stage of the spectrometer may undergo a nonneutralizing collision with a residual vacuum background particle in which energy will be lost. If the final stage is a magnetic sector, this energy-deficient ion will appear at a mass lower than its actual mass. If the final stage is an electrostatic sector, the energy deficient ion will not reach the detector at all.

B.2.1.5 Slit Widths and Resolution Resolution is defined as

R = m / ∆m, (9) the m / z measured divided by the difference of m / z to be separated. A Q-ICP-MS is operated in unit mass resolution, whereas a sector field provides the possibility of using the slit width to control resolution. A double focusing sector field MS in reverse Nier-Johnson geometry is displayed in Fig. B.2.1.5-1. At low resolution (R = 300), the ion beam is narrower than the slit. Thus, it is possible to obtain "flat-topped" peaks, which provide best precision for fast scanning in peak hopping mode. At higher resolution (up to R = 10 000), the slits are narrower than the ion beam.

23 Part B: Fundamentals

Therefore, measurements in high resolution are always at the expense of signal intensity and precision, which stems from the difficulty to measure a triangular peak exactly.

Figure B.2.1.5-1 Double focusing magnetic-sector mass analyzer in reverse Nier-Johnson-geometry, adapted from reference 40. MA = Magnetic mass analyzer; ESA = Electrostatic mass analyzer; B = Magnetic field.

B.2.2 Laser Ablation Inductively Coupled Plasma Mass Spectrometry (LA-ICP-MS) The coupling of laser ablation to ICP-MS is a powerful technique for the direct analysis of solid samples and was pioneered already by Gray.115 Nowadays, it is established for the analysis of bulk materials,116 geological,117 and biological28, 52 samples.27 The most widely used lasers are solid phase quintupled Nd:YAG (213 nm), quadrupled Nd:YAG (266 nm), and excimer ArF (193 nm) lasers at ns pulse duration. During the ablation process, laser photons are focused onto the surface of the sample. The laser beam is directed through a wavelength-transparent quartz glass window of the ablation chamber and interacts with the sample. In the resulting plasma of atoms, ions, and particles an aerosol is formed. The laser-generated aerosol is subsequently transported to the ICP by a carrier gas flow, where it is vaporized, atomized, and ionized. Finally, the ions can be analyzed by a mass spectrometer.

24 BAM-Dissertationsreihe Part B: Fundamentals

The use of He as a carrier gas is preferable over Ar due to an improved particle transport, resulting in an increase in sensitivity,118 and the reduction of signal spikes:119 The high thermal conductivity of He suppresses the coalescence of particles into larger ones (> 200 nm diameter) by efficiently removing energy from the ablation site.119 Furthermore, He does not efficiently transport larger (heavier) particles since it is lighter than Ar.119 The resulting detection efficiency in He is by a factor of up until 5 higher than in Ar, and this effect is independent of laser wavelength and pulse duration.120

The main problems in laser ablation arise from elemental fractionation, i.e. the occurrence of non-stoichiometric effects owing to the preferential ablation, transport, vaporization, atomization, or ionization of one analyte over another.121, 122 The experimental parameters applied for LA-ICP-MS influence the composition of the aerosol and hence, have been widely investigated in order to reduce or eliminate elemental fractionation, albeit the complex interdependency of all factors involved is not fully understood.123 The analysis of brass and the resulting Cu / Zn ratios are a good indicator of non-stoichiometric effects. The results of these studies indicate that particle size distributions have a major impact on the degree of fractionation.124-126 Upon the removal of larger particles from the carrier gas stream, less fractionation was observed since smaller particles are vaporized and ionized at higher efficiency.125 Furthermore, a Cu enrichment was observed for larger particles (> 100 nm), and Zn enrichment was found on smaller particles. However, large particles do not ionize completely, resulting in lower Cu / Zn ratios, and Zn deposition (owing to the lower melting point compared to Cu) leads to higher Cu / Zn ratios compared to the certified values. Nevertheless, the combination of various sources of elemental fractionation might finally result in an agreement between certified and measured values, since the underlying effects might compensate each other.124

Generally, the occurrence of elemental fractionation limits quantitative analysis in LA-ICP-MS for non-matrix matched calibration, and there is still a lack of reference materials for the majority of samples.127 However, with a careful optimization of all parameters involved, elemental fractionation is no problem for bulk analysis if equilibrium conditions are achieved with a few laser shots only.123 For other applications, it is recommended to apply a laser fluence ‘well above the ablation

25 Part B: Fundamentals threshold of the respective sample’, and low repetition rates for metallic samples to ‘keep the volume of melt small’.123

Improvements were also observed for fs-LA, which stem from differences in the ablation mechanism. The fs-pulse duration is shorter than many fundamental time- scales (phonon vibrations) and hence, the ablation process is non-thermal, which reduces elemental fractionation.127 Finally, the laser ablation chamber has significant influence on precision and sensitivity of the measurement. Thus, the cells are designed for minimal aerosol loss (around 1 % at atmospheric pressures),128 maximum aerosol density (fast washout times), and minimum signal dispersion. The position of gas inlet and outlet directly influence dead volume and washout times of the ablation cell, which is particularly important for the analysis of transient signals. Furthermore, signal intensity should be independent of the sample position in the ablation chamber.

B.3 DNA Microarrays Microarray analysis allows mRNA expression profiling for hundreds or thousands of genes in a single experiment and is therefore a tool widely used in functional genomics.129, 130 A DNA microarray consists of a glass slide with spots of immobilized oligonucleotide strands on the surface. When the microarray is incubated with a test sample, a snapshot of active genes is created. In what previously would have taken years, the high throughput capabilities allow the screening of a whole genome in a short period of time.

The DNA segments representing the collection of genes under investigation are amplified by polymerase chain reaction (PCR), and the resulting DNA probes are spotted on a coated microscope glass slide. Several functionalities are commercially available; one example being amino-modified DNA probes, which are covalently linked to an epoxy-coated glass slide, as illustrated in Fig. B.3-1. There are two options for microarray printing: (i) the sample is loaded onto a pin, and a small volume, typically a few nano- to picoliters, is transferred to the substrate by physical contact, (ii) the sample is loaded into a piezoelectric capillary, and an electric current is used to jet a small volume onto the glass slide without physical contact. In a microarray experiment, gene expression is determined by co-hybridization of

26 BAM-Dissertationsreihe Part B: Fundamentals fluorescently labeled cDNA probes from mRNA sources, which stem from the cellular phenotypes of interest.131 The resulting fluorescence intensity is measured by a confocal laser scanner, and the relative expression levels can be assessed by the ratio of which each target hybridizes to an individual microarray spot.

However, quantification is not feasible by this technique since the effectiveness of the fluorescent tag is dependent on its vicinity, which includes the sample itself and the substrate surface. Therefore, incalculable quenching effects might affect quantification. Furthermore, multiplex approaches might be hampered by spectral overlap of fluorescent tags.132 On the contrary, the application of elemental tags with subsequent ICP-MS detection provides multiplex capabilities due to a variety of stable tags, which are not influenced by characteristic features of its immediate environment, and which can be analyzed without spectral interferences.23, 47 Furthermore, quantification in ICP-MS can be performed at a large dynamic range and high sensitivity, if suitable standards are available.

In this work, a short primer sequence was employed for a proof-of-principle experiment for microarray detection by LA-ICP-MS. The target DNA was immobilized on the microarray surface as displayed in Fig. B.3-1, and a gold nanoparticle labeled probe DNA was used for signal amplification.

Figure B.3-1 Printing of amino-modified oligonucleotide, which functions as a target DNA, on epoxy-functionalized microarray substrate. The hybridization with labeled probe DNA follows in the next step. Only those probes will bind and result in a detectable signal, which are complementary to the target sequence.

B.4 Immunochemistry Immunoassays are routinely employed in biochemistry and medical diagnostics due to their capability of fast identification and quantification of biomolecules.133

27 Part B: Fundamentals

The specificity achieved is highly dependent on the antibodies selected for analysis. Antibodies are serum proteins produced in a vertebrate’s immune system by B-lymphocytes and plasma cells as a response to foreign target molecules (‘antigens’). The type of antibody used in immunoassays is immunoglobulin G (IgG), which accounts for 75 % of all serum immunoglobulins.85 It consists of two identical heavy chains (H) of 50 kDa, and two identical light chains (L) of 24 kDa.4 Light and heavy chain, as well as their identical counterpart, are linked by non-covalent forces and disulfide bonds (see Fig. B.4-1. The N-terminal domains of each light and heavy chain are highly variable, and represent the two antigen-binding sites. The C-terminal domains are constant in their amino acid sequence. They are responsible for antibody binding to a cell surface, and also for immobilization on the microtiter plate in immunoassays. Upon papain digestion, the antibody dissociates into three parts: two Fab fragments (antigen binding), and one Fc fragment (crystallizable region).

Figure B.4-1 Scheme of immunoglobulin G (IgG). Light (light blue) and heavy (dark blue) chains are connected by disulfide bonds. Parts with variable amino acid sequence, which represent the antigen binding sites, are ruled. VH: variable heavy; VL: variable light. Upon papain digestion, the antibody dissociates into three parts: two Fab fragments (antigen binding), and one Fc fragment (crystallizable region). Adapted from reference 134.

28 BAM-Dissertationsreihe Part B: Fundamentals

For antibody production, a substance triggering the immune response (‘immunogen’), is injected to a host animal. By means of the Fab fragment, antibodies recognize a specific steric pattern (‘epitope’) on the surface of an antigen. Small molecules below 1500 Da, which do not trigger an immune response (‘haptens’),135 but can be conjugated to a carrier protein for immunization.136 Polyclonal antibodies are obtained from serum and therefore originate from different B-lymphocytes, causing heterogeneous specificity for a variety of epitopes. The reproducible production of homogeneous, and thus highly specific monoclonal antibodies is feasible by the production of genetically identical hybridoma cell lines.135

B.4.1 Immunoassays The first description of an antibody carrying a label for subsequent detection was published by Coons in 1941.137 He introduced the coupling of fluorescein by an isothiocyanate derivate. However, fluorescent labels suffered from quenching until long-lived fluorescence in rare earth elements was discovered,138 which was exploited for immunoassays about 40 years later.139 The first clinical immunoassay, making use of the specificity of antigen-antibody reactions, was developed for the determination of insulin in human blood (Yalow and Berson, 1960).140 Radioactive labels were applied for detection, but the accompanying health hazards led to the development of enzymatic labels by the same group,8 who initiated the era of enzyme-linked immunosorbent assays (ELISA). The solid phase technology enabled automation and thus, a high sample throughput. Several formats are available and are described in textbooks.4 Due to the immobilization on a solid phase, diffusion, which is inherently slow, is the limiting factor for antibody reactions. To overcome this problem, antibodies can be conjugated to magnetic beads for example, so that the antibody reaction takes place in solution, followed by immobilization through the application of a magnetic field.133

In this work, an indirect competitive assay was employed and is illustrated in Fig. B.4.1-1: An analyte-protein conjugate is immobilized on a microtiter plate, and competes with free analyte for a limited number of antibody binding sites. The secondary antibody is labeled with an enzyme (horseradish peroxidase), which catalyzes a chromogenic reaction; in this case the oxidation of TMB.

29 Part B: Fundamentals

The absorbance is measured in a microtiter plate spectrometer, which shows high signals for low analyte concentrations owing to the competitive assay format.

Figure B.4.1-1 Indirect competitive immunoassay for OTA detection. a Coating of analyte-BSA conjugate followed by incubation of sample and primary antibody; Washing removes unbound antibody. The next step involves incubation with enzyme-conjugated secondary antibody, which b catalyzes a chromogenic reaction, e.g. the oxidation of TMB. The resulting color development is detected by photometry.

Competitive immunoassay standard curves display a sigmoidal response when plotted on a log/linear graph and data processing is usually accomplished by a 4-parameter log fit with A: minimum response, D: maximum response, C: point of inflection, and B: slope at point of inflection.141

30 BAM-Dissertationsreihe Part B: Fundamentals

(10)

The limit of detection (LOD) of the assay can be determined as 85 % of the signal intensity, which corresponds with the 3s definition assuming a 5 % relative standard deviation.142 Due to the sigmoidal calibration function, error distribution of the concentration values is heteroskedastic. This leads to an increase in uncertainty for very high and very low values of the standard curve. Ekins proposed the precision profile for determination of the quantification range, which was applied in this work.143

B.4.2 Immunohistochemistry (IHC) Immunohistochemistry is a method for the specific detection of proteins in tissue sections and has become one of the most powerful techniques in cancer diagnostics to assess the level of expression of a tumor marker.85 In the direct format, the label is directly conjugated to a primary antibody; in the indirect format, a labeled secondary antibody is employed for signal amplification. Immunofluorescence was the first detection method employed for IHC.144 Furthermore, the specific antigen-antibody reaction can be visualized by the use of enzymatic or electron-dense (e.g. ferritin, gold nanoparticles) labels.134 However, an enzyme-labeled secondary antibody is commonly applied for signal amplification, since the resulting stained tissue sections can be studied by light microscopy.92 A variety of enzymes has been employed for IHC, and peroxidase or alkaline phosphatase are only two examples. Graham and Karnovsky proposed the visualization of peroxidase by oxidation of 3,3’ diaminobenzidine (DAB), which results in a brown stain.145 In contrast to ELISAs, the dye produced after enzymatic reaction is insoluble and hence, is absorbed on the tissue in vicinity of the antibody binding site. For additional morphologic studies of the investigated tissue, it is usually counterstained with hematoxylin, which imparts a blue color to basophil cell structures, e.g. chromatin of cell nuclei.

B.5 Gel electrophoresis Electrophoresis describes the migration of charged particles in an electric field in agarose or polyacrylamide gels, and is widely used for the separation of macromolecules. Agarose gels are suited for the separation of molecules over

31 Part B: Fundamentals

500 kDa like DNA or RNA, whereas polyacrylamide gels are usually employed for the separation of proteins typically in the range of 10–200 kDa for a gradient gel, wherein pore sizes continuously increase in the gel.4 A gel electrophoresis (GE) experiment requires a high voltage generator, two electrodes, and a temperature-controlled separation chamber, in which the gel is embedded between two glass plates. If a voltage is applied between the two electrodes, an electric field is created, and charged molecules will migrate to the electrode of opposite charge. In sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separation is based on the high affinity of SDS to proteins (1.4 g SDS bind to 1 g protein), and thus the net charge is dominated by the negative charge of SDS, being proportional to protein size. Secondary and non-disulfide-linked tertiary structures are denatured by SDS. Additionally, disulfide bonds are disrupted by heating the sample to 90 °C in the presence of reducing agents like beta-mercaptoethanol or dithiothreitol. The negatively charged molecules migrate to the anode (+) during electrophoresis. The migration of particles is proportional to their charge and the electric field, and is inversely proportional to the particle radius and viscosity of the gel. Owing to differences in charge and radius, biomolecules have a characteristic migration velocity and form discrete bands; biomolecules with a large radius migrate slowly.

The discontinuous electrophoresis, developed by Tulchin et al.,146 is a technique capable of focusing and concentrating the sample in the gel before separation and thus, creates sharp bands. This method is characterized by the different pH of the buffers used, and the different pore sizes of the two gels employed, namely the stacking gel and the resolving gel. The stacking gel (pH 6.8) focuses the sample in the gel before separation occurs in the resolving gel (pH 8.8). At the border of stacking and resolving gel, friction is suddenly increased, which generates a reduction of migration velocity for all biomolecules, causing focusing and thus, sharp bands. Subsequently, separation is performed in the resolving gel. The combination of SDS-PAGE with isoelectric focusing (IEF) enables a separation of proteins in two dimensions (2D-GE) by their isoelectric point (pI), and size. This process is illustrated in Fig. B.5-1.

32 BAM-Dissertationsreihe Part B: Fundamentals

C G C t = 0 AABH B H F EED D F G

low pH gradient high

A B CCE F G H t > 0 B A DDE F G H

second dimension high MW AA FF CC

EE HH low MW

Figure B.5-1 Two-dimensional gel electrophoresis with isoelectric focusing, and SDS-PAGE separation. MW: molecular weight. Adapted from reference 147.

During IEF, separation occurs according to the proteins’ pI. The charge of a protein is pH dependent and at a characteristic pH the net charge of the protein is zero, and it is no longer influenced by the electric field. A gel with immobilized pH gradient (IPG) is used for isoelectric focusing. The IPG strip is laid parallel to the cathode (-), and in the second dimension the negatively charged proteins migrate to the anode during SDS-PAGE. Following separation, protein spots are commonly visualized by Coomassie Brilliant Blue (CBB)148 or silver,149 albeit a variety of staining techniques is available.54 Nowadays, mostly colloidal CBB150 is used with a low concentration of free dye, which reduces background staining of the gel. The dye sticks to the amino groups of the proteins by electrostatic and hydrophobic interactions at acidic pH. The limit of detection is approximately 10 ng, with a linearity range of 103.54 During silver staining, protein-bound ions are reduced to metallic silver. This technique is more sensitive than CBB staining (LOD approximately 1 ng), with a linearity range of 102.54 Both staining methods are compatible to MS methods for subsequent protein identification.

Part C: Experimental

Part C: Experimental The vast majority of analyses presented in this work were performed by ICP-MS, which requires a careful optimization of instrumental parameters and operating conditions. For example, the optimization of gas flows, ion lens settings, RF power, and torch position was performed daily, using a solution containing 1 ng g-1 Na, In, U to assure a sensitivity of at least 1 200 000 cps for In and 1 800 000 cps for U in low resolution mode, and a RSD of ≤ 2 %. For LA-ICP-MS of biological samples, which were usually placed on microscopic glass slides into the ablation chamber, the ICP was tuned daily to achieve maximum ion intensity, while keeping the oxide ratio (ThO / Th) below 1 % during ablation of a microscopic glass slide, and a RSD of Th ≤ 5 %. Furthermore, the He carrier gas flow was individually optimized for each ablation chamber. Tygon tubing (Compagnie de Saint-Gobain, France) with an inner diameter of 3.2 mm was used for aerosol transport. The laser ablation parameters applied for analysis, such as laser energy, spot size, scan speed, and repetition rate, were optimized daily on a parallel sample to assure high sensitivity with concomitant high spatial resolution. For imaging applications, the ICP-MS was synchronized with the LA unit in external triggering mode.

Data processing for imaging is described in the following: Single line scans, which contain elemental intensity time profile data, were exported to Origin 8.5 to calculate surface plots by converting both the number of line scans and the scan speed into a millimeter scale. Elemental intensities were color coded in a way that high intensities are shown in red and low intensities are shown in blue. Corresponding images from a simultaneous measurement can be merged by the software ImageJ.

Two commercial ns laser ablation systems at 213 nm wavelength, the LSX-213 from CETAC (Omaha, Nebraska, USA) for the ablation of gels, and the New Wave 213 from esi (Portland, Oregon, USA) for the ablation of thin tissue sections, were employed in this work. Furthermore, an ArF excimer laser at 193 nm (GeoLasC, Coherent, Göttingen, Germany) was applied for microarray detection.

35 Part C: Experimental

C.1 Immunoassay As already discussed in section B.1.3, the detection power of ICP-MS was compared to a conventional photometric detection in an immunoassay for OTA determination in wine, which was optimized in this work. An indirect competitive format was applied in this work and is illustrated in Fig. B.4.1-1. The specific binding of antigens with antibodies is the basis of an immunoassay. In this format, an analyte-protein-conjugate is immobilized on the well plate and competes for antibody binding sites with unbound analyte. A washing step removes the latter.

Figure C.1-1 Indirect competitive immunoassay for OTA determination. Photometric detection: Addition of TMB, which induces a color development; ICP-MS detection: Acid digestion of gold nanoparticle labeled secondary antibodies, and addition of Ir as internal standard for liquid ICP-MS measurements.

Secondary antibodies are used for signal amplification and bind to the analyte-antibody-complex on the well plate. Thus, a high analyte concentration results in a weak signal. The secondary antibody was labeled with an enzyme (horseradish peroxidase) for photometric detection, whereas a gold nanoparticle conjugate was employed for ICP-MS detection, as displayed in Fig. C.1-1. For color development and subsequent photometric detection, TMB with hydrogen peroxide was applied as a substrate for horseradish peroxidase.

36 BAM-Dissertationsreihe Part C: Experimental

C.1.1 Materials Transparent, 96 flat-bottom wells microtitre plates with high protein binding capacity were purchased from Nunc (Wiesbaden, Germany). Incubation was performed on a Titramax 101 plate shaker (Heidolph, Schwabach, Germany) set to 750 rpm. Washing was accomplished with an automatic 96-channel plate washer (ELx405 Select BioTek Instruments, Bad Friedrichshall, Germany). A microplate spectrophotometer Spectramax Plus384 (Molecular Devices, Sunnyvale, USA) controlled by SoftMax Pro software (v 5.2, Molecular Devices) was used for photometric detection. The absorbance was measured at 450 nm, using 650 nm as a reference wavelength.

C.1.2 Buffers and Chemicals All solutions were prepared using ultrapure water (> 18 MΩ; Milli-Q water purification system, Millipore, Schwalbach, Germany), unless stated otherwise. Ultrapure water is referred to as Milli-Q water in the following. Buffer salts (sodium carbonate, sodium hydrogencarbonate, monopotassium phosphate, dipotassium phosphate, sodium chloride, monosodium phosphate, disodium phosphate, potassium dihydrogen citrate) were of Fluka "ultra” quality (Sigma-Aldrich, Taufkirchen, Germany). Sodium hydroxide (Biochemika Ultra, >98,0%), hydrogen peroxide 30% Trace select, tetrabutylammonium borohydride (TBAB) (purum), and potassium sorbate (p.a.) were also from Fluka. Tris(hydroxymethyl)-aminomethan (p.a. for buffer) was purchased from Merck (Darmstadt, Germany), and hydrochloric acid (reagent grade, 37%) was supplied by Sigma-Aldrich. 3,3’,5,5’-Tetramethylbenzidine (TMB), research grade, and Tween™ 20 (pure), were purchased from Serva (Heidelberg, Germany). N,N-Dimethylformamide (anhydrous, 99.8%) was from Sigma Aldrich, and sulphuric acid (95-97% p.a.) was from Mallinckrodt Baker (Griesheim, Germany).

37 Part C: Experimental

Table C.1.2-1. Buffers used for OTA immunoassay. All buffers were prepared in Milli-Q water if not stated otherwise. The pH was adjusted with NaOH or HCl.

Solution name Composition Carbonate buffer 15 mM sodium carbonate 35 mM sodium hydrogencarbonate pH 9.60

Phosphate buffer 40 mM monopotassium phosphate 375 mM dipotassium phosphate 1 mM potassium sorbate pH 7.60

Washing buffer 0.05 % (v/v) Tween™ 20 in 1:60 phosphate buffer pH 7.60

Tris buffer 100 mM tris-(hydroxymethyl)-aminomethane 150 mM sodium chloride pH 7.50

PBS buffer 10 mM monosodium phosphate 70 mM disodium phosphate 130 mM sodium chloride pH 7.60

Substrate buffer 220 mM potassium dihydrogen citrate 0.5 mM potassium sorbate pH 4.00

TMB solution 40 mM tetramethylbenzidine 8 mM tetrabutylammonium borohydride In N,N-dimethylacetamide

Substrate solution 550 µL TMB solution 3 mM hydrogen peroxide In 22 mL substrate buffer

38 BAM-Dissertationsreihe Part C: Experimental

Casein sodium salt (lot 045K0159), albumin from bovine serum (BSA, 96 %, A 9418, Batch#: 084K0578), and OTA–BSA 5 mg mL-1 were purchased from Sigma-Aldrich. Ochratoxin A (lot OC020) was from Fermentek, Israel.

Iridium standard was diluted from 1 g L-1 stock solution (Merck, Darmstadt, Germany). Nitric acid and hydrochloric acid were purified in-house prior to use by sub-boiling distillation of reagent grade feedstock in a quartz still. Digestion was achieved with a 1:3 mixture of concentrated nitric acid: hydrochloric acid, which was further diluted in Milli-Q water for use on the well plates.

C.1.3 Antibodies Mouse monoclonal anti-OTA antibody was kindly provided by Chung et al.,151 Division of Applied Life Science, Graduate School of Gyeongsang National University, Chinju, Gyeongnam 660-701, South Korea, 0.5 mg mL-1). Anti-mouse IgG (whole molecule)-Peroxidase, goat, 0.8 mg mL-1 (lot 067K6010) was purchased from Sigma-Aldrich. Gold labeled anti-mouse IgG (H+L) System 40 nm, goat, 0.1 mg mL-1 (lot 090028) was from KPL (Gaithersburg, MD, USA).

C.1.4 Wines White wine “Captain Selection”, Australia, 2005 and red wine “Mundelsheimer Schalkstein Trollinger”, Germany, 2005 were purchased from a local supermarket. The red wine reference material (ERM®-BD476) was from BAM Federal Institute for Materials Research and Testing.

C.1.5 Immunoassay Procedure Each standard sample was prepared in triplicate wells, wine samples in quadruple wells. The assay was performed at room temperature. After each incubation step, microtitre plates were sealed with Parafilm and deposited on a plate shaker.

The transparent high-binding microtitre plates were coated with OTA–BSA conjugate in carbonate buffer (250 µg L-1, 100 µL, 18 h). The wells were then blocked with 0.1 % casein in carbonate buffer (200 µL, 30 min). OTA standards and wine samples were prepared in 100 mM Tris buffer (pH 7.5) / 0.1 % casein. After washing

39 Part C: Experimental three times with washing buffer, two incubation steps were conducted: the first involved a pre-incubation with OTA standard or wine (50 µL, 10 min), directly followed by incubation of the primary mouse monoclonal anti-OTA antibody in PBS / 0.1 % casein (50 µg L-1, 50 µL, 1 h). For the second incubation step, anti-mouse IgG conjugated with either gold nanoparticles or with horseradish peroxidase were applied (500 µg L-1 in manufacturer’s dilution buffer or 80 µgL-1 in PBS / 0.1 % casein, respectively, 1 h). A washing step followed each incubation step. For detection with the Spectramax Plus384, substrate solution was applied (100 µL, 20 min). The reaction was stopped by 1 mol L-1 sulphuric acid (50 µL). Gold labeled secondary antibody was dissolved in 20 % (v/v) digestion acid (200 µL, 5 h). A 100 µL volume of the sample was added to 100 µL of a 1 µg L-1 iridium standard solution (diluted from 1 g L-1 stock solution, Merck, Darmstadt, Germany) and measured by ICP-MS. Data processing was accomplished with the 4-parameter log fit.141

C.1.6 ICP-MS Detection Detection of digested gold nanoparticles was accomplished on a sector field instrument (Element XR, Thermo Fisher Scientific, Bremen, Germany). The instrument was equipped with a Scott type spray chamber and a PFA self aspirating nebulizer operating at 50 µL min-1. ICP-MS measurements were performed with 15 scans, usually achieving a relative standard deviation below 2 % for a single measurement.

40 BAM-Dissertationsreihe Part C: Experimental

Table C.1.5-1 Instrumental parameters and operating conditions of the ICP-MS.

Instrumental parameters ICP-MS Element XR -1 Sample introduction PFA self aspirating nebulizer 50 µL min ; Scott-type spray chamber RF plasma source [W] 1350 Plasma gas [L min-1] 15 Auxiliary gas [L min-1] 0.8 Nebulizer gas [L min-1] 1.06 Sampler and skimmer cone Nickel Data acquisition

7 24 65 109 191 193 197 238 Isotopes monitored Li, Mg, Cu, Ag, Ir, Ir, Au, U Integration time [ms] 600 Runs / Passes 3 / 5 Total time [min:s] 01:20

Iridium was used as an internal standard for gold due to their close atomic numbers and ionization potentials. Thus, it is expected that they show similar responses during ICP-MS measurements. To quantify the gold content, the 197Au / (191Ir + 193Ir) ratio was used. The instrumental parameters and operating conditions of the ICP-MS are given in Table C.1.5-1. The isotopes monitored covered a broad mass range to ensure a precise magnet settling. The resulting longer integration time was not critical here due to the continuous liquid sample introduction.

C.2 DNA Microarrays By means of a 16mer primer sequence as a target DNA, and a 16mer probe DNA labeled with gold nanoparticles, a proof-of-principle experiment was designed for LA-ICP-MS-based microarray detection. As already discussed in section B.3, this could provide a quantification approach, which is independent of the vicinity of the probe.

41 Part C: Experimental

C.2.1 Chemicals and Materials Chemicals used for immunoassays and microarrays were from the same manufacturers, if not stated otherwise. Trisodiumcitrate (Ultra for molecular biology, lot 134105252308071) and hydrochinon ≥ 99 % (lot 1396433348061239) were purchased from Fluka. Silver nitrate (99.9999 %) and polyethylene glycol (4600 average molecular weight, lot 11022 PD-427) were from Sigma-Aldrich. SuperBlock T20 (PBS) Blocking Buffer (lot JG121073) was obtained from Thermo Scientific (Piercenet, Rockford, USA). All solutions used for microarray processing are specified in Tables C.2.1-1 and C.2.1-2.

42 BAM-Dissertationsreihe Part C: Experimental

Table C.2.1-1 Microarray buffers. All buffers were prepared in Milli-Q water. The pH was adjusted with NaOH or HCl.

Solution Composition Print buffer 0.4 M disodiumphosphate 0.04 M monosodiumphosphate pH 8.0 Blocking buffer 0.1 M tris-(hydroxymethyl)-aminomethane pH 8.5 20×SSC 3 M NaCl 0.3 M trisodiumcitrate pH 7.0 Wash buffer I (DNA) 4×SSC 0.1 % (v/v) sodium dodecyl sulfate PBS buffer 10 mM monosodium phosphate 70 mM disodium phosphate 130 mM sodium chloride pH 7.60 -1 Labeling buffer 10 mg mL PEG : SuperBlock Plus buffer; 1 : 1 Phosphate buffer 40 mM monopotassium phosphate 375 mM dipotassium phosphate 1 mM potassium sorbate pH 7.60 Wash buffer II 0.05 % (v/v) Tween™ 20 in 1:60 phosphate buffer pH 7.60 Citrate buffer 15 mM potassium dihydrogen citrate 0.15 mM potassium sorbate pH 3.8 Hydrochinone solution 0.333 g hydrochinone in 100 mL citrate buffer Silver solution 0.7 % (w/v) silver nitrate in Milli-Q water Silver enhancer Hydrochinone solution : silver solution; 9 : 1

Table C.2.1-2 Hybridization buffer.

Stock solution Volume [µL] Final conc. in 20 µL 20 x SSC 4 4 x SSC 10 mg mL-1 PEG 2 1 mg mL-1 PEG 1 M Tris pH 7.5 1 50 mM Tris, pH 7.5 1 % SDS 4 0.2 % SDS Oligonucleotide 100µM 4 Oligonucleotide 20 µM 5 Milli-Q water

43 Part C: Experimental

A single stranded, 16mer oligonucleotide with a (CH2)12-linker and an amino modification at the 5’-ending (5’-C12 NH2-CACAATTCCACACAAC) was immobilized on epoxy-coated microarray slides (75 mm × 25 mm, Austrian Research Center, Vienna, Austria) either by pipetting or by use of a microarray printer implementing solid ceramic pins (Bio Odyssey Calligrapher 2.0 Mini Arrayer, BioRad, Munich, Germany). Hybridization was performed with a single stranded, 16mer target oligonucleotide with a triethyleneglycole-linker and a biotin modification at the 5’-ending (5’-Bio-GTTGTGTGGAATTGTG), or with a Cy3 modification at the 5’-ending for fluorescence detection. All oligonucleotides were purchased from Tib MolBiol (Berlin, Germany) in lypophilized form and were resuspended in Milli-Q water to a concentration of 100 µM (storage at 4 °C). A streptavidin-gold conjugate (Streptavidin from Streptomyces avidinii conjugated with 10 nm gold nanoparticles, lot 027K6090, Sigma Aldrich, Germany) was employed for labeling of the biotin modified oligonucleotide.

Fluorescence was detected at Scienion AG (Berlin, Germany) by a microarray scanner at 532 nm and 10 µm pixel size (GenePix 4000B, Molecular Devices, CA, U.S.A.), and gold labeled oligonucleotides were analyzed by LA-ICP-MS. The optimization of the microarray assay including printing, hybridization, blocking, and labeling was performed at BAM, and LA-ICP-MS analyses were conducted with the LSX-213 (CETAC) and the SuperCell (New Wave). The final measurements were performed at ETH Zürich in collaboration with Detlef Günther and Mattias Fricker on an ELAN 6100 DRC+ (Perkin Elmer, MA, USA) coupled to an ArF excimer laser at 193 nm (GeoLasC, Coherent, Göttingen, Germany). The samples were inserted into a laser ablation cell (see Fig. C.2-1) designed at ETH Zürich,152 which permits the ablation of large samples (available space for samples 230 mm × 34 mm × 16 mm, L × W × H), or many small samples, e.g. up to three microarray slides (76 mm × 26 mm × 1 mm, L × W × H). Between 8 and 10 microarray spots (distance 2 mm) could be ablated in x-direction without moving the sled. The operating conditions of the LA-ICP-MS are specified in table C.2.1-3. The single pulse ablation was tested on NIST 610 under these conditions, and the 197Au isotope was monitored. According to GeoReM, NIST 610 has an Au content of 23 µg g-1.153

44 BAM-Dissertationsreihe Part C: Experimental

Figure C.2-1 Laser ablation chamber used for microarray analyses. The He carrier gas flow enters the ablation chamber through two gas inlets, and the sample aerosol leaves the chamber through a gas outlet, which is centered on the edge of the ablation window. The ablation of large samples is permitted by this chamber, and the sample is positioned below the ablation window by a manually movable sled. During laser ablation, the position of the cell can be computer-controlled by a xy-stage.

C.2.2 Sample Preparation The amino modified oligonucleotide was diluted in print buffer for immobilization to a concentration of 20 µM. Heated buffers had a temperature of approximately 37 °C.

Immobilization was performed by pipetting and subsequent incubation in a humidity chamber, or by a microarray spotter at 65 % humidity control over night, followed by washing the slide with heated DNA wash buffer three times. Slides can be stored at room temperature (RT) at this point. For further processing, slides were blocked with heated blocking buffer for 15 minutes, and rinsed three times with heated DNA washing buffer. Hybridization was carried out overnight at different probe oligonucleotide concentrations in a hybridization chamber (Arrayit, Sunnyvale, CA, USA) to prevent drying of the sample, followed by rinsing with heated DNA wash buffer.

45 Part C: Experimental

For blocking of the substrate prior to labeling with gold nanoparticles, different strategies were pursued: The dilution of the streptavidin-gold (SA-Au) conjugate with 10 mg mL-1 polyethylene glycol (PEG) in Milli-Q water, or in PBS buffer, or in wash buffer II (SA-Au : PEG = 1:10) did not reduce nonspecific binding during labeling. Even for higher dilutions (1:100 1:1000), negative controls were indistinguishable from positive controls. The use of O-(2-Mercaptoethyl)-O'-methyl-hexa (ethylene glycol) (mPEG-thiol) was not successful either (mPEG-thiol : SA-Au : wash buffer II = 1:10:500). Finally, the optimization resulted in the following conditions: Before labeling, slides were incubated with 10 mg mL-1 PEG solution for 10 minutes to block the glass surface. Labeling was performed in a humidity chamber (Arrayit) for one hour with a 1:1 mixture of PEG solution and SuperBlock T20 Buffer. The dilution of SA-Au was varied from 1:10, 1:100, and 1:1000. Best results were achieved by a 1:100 dilution of streptavidin-gold conjugate in labeling buffer. As last step, the slide was rinsed three times with wash buffer II. For visualization of microarray spots, silver enhancement was applied. The microarray slide was incubated with silver enhancer solution at RT on a shaker for not more than 20 minutes and finally rinsed with Milli-Q water. Slides can be stored at RT.

46 BAM-Dissertationsreihe Part C: Experimental

Table C.2.1-3 Instrumental parameters and operating conditions for microarray analysis by LA-ICP-MS.

Laser ablation

Laser 193 nm ArF excimer

-2 Fluence [J cm-2] 6.3–7.9 J cm

Laser spot size [µm] 160 square

Ablation mode Single pulse (The location on the slide was computer controlled, and the movable sled was manually adjusted between the slides.)

ICP-MS

+ Instrument ELAN 6100 DRC

RF power [W] 1400

Carrier gas, He [L min-1] 1.0

Nebulizer gas, Ar [L min-1] 0.77

Auxiliary gas, Ar [L min-1] 0.8

Plasma gas, Ar [L min-1] 17.5

197 Dwell time [ms] 50 ( Au) 1 (137Ba)

Data acquisition Pulse counting (fast settling time)

C.3 Immunohistochemistry Immunohistochemistry is a method employed in cancer diagnostics. An antibody reacts with a tumor marker, e.g. an oncoprotein, with high selectivity. Commonly an indirect method is applied for IHC, using a primary antigen-specific antibody and a second incubation with an enzyme-labeled secondary antibody for signal amplification.92 Primary antibodies, commonly applied in IHC, were labeled with lanthanides via a DOTA-linker for LA-ICP-MS detection.

47 Part C: Experimental

C.3.1 Labeling of Primary Antibodies The labeling of antibodies involves (i) the purification of the antibody if necessary, (ii) the production of the metal chelate, and (iii) the antibody labeling. The purification of antibodies was optimized in this work, whereas the production of the metal chelate, and the antibody labeling were subject to slight modifications of a method described by Waentig et al.57

C.3.1.1 Chemicals All solutions used for the labeling of primary antibodies are specified in Table C.3.1.1-1. Buffer chemicals were from the same manufacturers as stated in section C.1, if not mentioned otherwise. Tetrabutylammonium acetate (TBAA) was purchased by Sigma-Aldrich (Taufkirchen, Germany), glycine was from Applichem

(Darmstadt, Germany), and lanthanide chlorides (HoCl3, TbCl3, TmCl3) were obtained from Acros Organics BVBA (Geel, Belgium).

C.3.1.2 Purification of Antibodies Anti-Her 2 (Dako Deutschland GmbH, Hamburg, Germany) and anti-MUC 1 (antikoerper-online, Aachen, Germany) were purchased in purified form, whereas anti-CK 7 (Dako Deutschland GmbH, Hamburg, Germany) was provided as cell culture supernatant by the manufacturer. Purification of anti-CK 7 was performed prior to labeling using a 1 mL HiTrap Protein G column (Amersham pharmacia biotech, Piscataway, NJ, USA). First, the column was conditioned with 10 mL sodium phosphate buffer, followed by adding 35 µg anti-CK 7 and rinsing of the column with another 10 mL of sodium phosphate buffer. The sample was then eluted with 5 mL glycine-HCl. Three drops each were collected in a UV transparent 96 well plate containing 150 µL of carbonate buffer to reach a final pH 7 for antibody stabilization. By measuring the absorbance at 280 nm using a microplate spectrophotometer Spectramax Plus384 (Molecular Devices, Sunnyvale, USA), antibody containing fractions were selected. The eluate was concentrated using a 30 kDa cut off Microsep Centrifugal Device (VWR, Darmstadt, Germany) and resumed in carbonate buffer for labeling. Protein concentration was measured at 280 nm with BSA as a calibration standard. Recovery of HiTrap Protein G column is approximately 70 %, and was tested with anti-lysozyme.

48 BAM-Dissertationsreihe Part C: Experimental

Table C.3.1.1-1 Solutions used for labeling of primary antibodies.

Solution Composition TBAA buffer 20 mM tetrabutylammonium acetate pH 5.5 -1 Lanthanide stock solution 10 nmol µL lanthanide (Ho, Tb, Tm) chloride in TBAA buffer -1 p-SCN-Bn-DOTA stock solution 4 mg mL p-SCN-Bn-DOTA in TBAA buffer Carbonate buffer 0.1 M sodium carbonate : 0.1 M sodium hydrogencarbonate = 1 : 10 pH 9.0

Sodium phosphate buffer 20 mM monosodium phosphate 8 mM disodium phosphate pH 7.0

Glycine-HCl buffer 0.1 M glycine pH 2.7

Tris buffer 0.1 M tris-(hydroxymethyl)-aminomethane pH 7.5

C.3.1.3 Production of Metal Chelate The macrocycle p-SCN-Bn-DOTA (Macrocyclics, Dallas, USA) was loaded with the lanthanide (Ln) of interest in a molar ratio of n[Ln3+]:n[SCN-DOTA] = 2:1 in TBAA buffer. The mixture was incubated for 1 h at 37 °C. Purification was accomplished by a solid phase extraction column (DSC-18, 3 mL, Sigma Aldrich, Deisenhofen, Germany), used in combination with a vacuum chamber. Column conditioning was performed with 2.5 mL acetonitrile and 3 mL TBAA buffer. The sample was then infiltrated on the column and washed by 5 mL TBAA buffer. Finally, the Ln-DOTA-complex was eluted in 3 mL 2:1 acetonitrile:TBAA buffer. The remaining acetonitrile was evaporated in a gentle stream of nitrogen.

49 Part C: Experimental

C.3.1.4 Antibody Labeling The antibody was labeled with an excess rate of the ligand n[SCN-DOTA(Ln)]:n[Ab] = 100:1 in carbonate buffer of 500 µL total volume. The reaction vessel was placed on a shaker for four hours. A PD Midi-Trap G-25 column (Sephadex, GE Healthcare, Munich, Germany) was used for purification according to the manufacturer’s instructions, and the labeled antibody was eluted in 1 mL 0.1 M Tris buffer. The sample was concentrated, using a 30 kDa cut off Microsep Centrifugal Device (VWR, Darmstadt, Germany). Centrifugal devices were incubated over night in 10 % glycerol to avoid nonspecific binding of the antibody to the filter membrane, according to the manufacturer’s instructions. After removal of the glycerol solution, the sample was loaded into the centrifugal device, preconcentrated to 300-500 µL by ultrafiltration, and washed three times with 800 µL Tris buffer. Anti-CK 7 was labeled at a higher excess rate of n[SCN-DOTA(Ln)]:n[Ab] = 275:1 to guarantee a ligand excess even in those cases where the purification is not sufficient and cell culture proteins remained in solution. Concentrations of labeled antibodies were measured in a Bradford Assay using the Bradford reagent from Sigma Aldrich (Taufkirchen, Germany). Absorption of the antibody-dye-complex was measured with a microplate spectrophotometer at 595 nm. BSA was used as a calibration standard.

C.3.2 Immunohistochemical Reaction Handling of the tissues was performed at HELIOS Klinikum Emil von Behring, Institut für Pathologie, Berlin.

C.3.2.1 Conventional IHC staining Formalin-fixed, paraffin-embedded (FFPE) tissue blocks of human breast cancer were sectioned using a conventional sliding microtome (Leica SM 2010R, Leica Microsystems, Wetzlar, Germany) with a thickness setting of 3 µm. The sections were mounted onto Superfrost Plus slides (Thermo Fisher Scientific, Braunschweig, Germany) for IHC staining. These slides have a special organic coating in order to fix tissue sections during IHC procedure. Thin sections for IHC staining were processed by the fully automated BenchMark XT slide preparation system (Ventana Medical Systems, Inc., Tucson, AZ, USA). The sequence of automated events for immunohistochemical analyses is specified in Table C.3.2.1-1. Paraffin was removed

50 BAM-Dissertationsreihe Part C: Experimental from tissue samples, followed by heat-induced epitope retrieval (for c-erbB-2 and mucin 1). Cytokeratin 7 was retrieved by protease. Tissues were then incubated with primary and secondary antibody, which are applicable to paraffin-embedded sections. The last step involves visualization of binding events.

Table C.3.2.1-1: Sequence of automated events for IHC analyses. All solutions used in combination with the BenchMark XT were from Ventana (Ventana Medical Systems, Inc., Tucson, AZ, U.S.A.), if not stated otherwise. Primary antibodies were diluted in antibody diluent.

Step Reagent Deparaffinization EZ prep buffer Rinsing Wash buffer Epitope retrieval For anti-CK 7: Protease 1 (20 min, RT) For anti-Her 2 and anti-MUC 1: Target retrieval solution pH 6 (20 min, 90 °C) Rinsing Wash buffer -1 Incubation with primary 3.3 µg mL monoclonal mouse anti-human antibody cytokeratin 7 (anti-CK 7, clone OV-TL-12/30), 30 min -1 0.8 µg mL polyclonal rabbit anti-human c-erbB-2 oncoprotein (anti-Her 2), 30 min -1 4.6 µg mL monoclonal mouse anti-human mucin 1 (anti-MUC 1), 30 min Rinsing Wash buffer -1 Incubation with secondary < 50 µg mL , 30 min antibody Rinsing Wash buffer Staining Ventana UltraView Universal Alkaline Phosphatase Red Detection Kit Rinsing Wash buffer Counter stain Hematoxylin II Rinsing Wash buffer Dehydration Graded alcohols and xylene Coverslipping

51 Part C: Experimental

C.3.2.2 IHC for LA-ICP-MS The FFPE tissues were sectioned at 5 µm for LA-ICP-MS measurements by a microtome and mounted onto Superfrost Plus slides. Thin sections were deparaffinized in xylene, rehydrated through a series of alcohols, and incubated in target retrieval solution pH 6 (Ventana Medical Systems) for 20 min at 90 °C. For the multiplex experiment, after rinsing with wash buffer, an aliquot (100 µL) of all diluted primary antibodies (~ 1 µg mL-1 anti-Her 2, anti-CK 7, and anti-MUC 1) at the same time was placed onto the sections for 3 hours. Incubation was performed in a hybridization chamber to prevent drying of the tissue. In the following step, tissue sections were rinsed with wash buffer to remove unbound antibodies, and dehydrated through graded alcohols prior to laser ablation. A series of graded alcohols consists of 70 %, 90 %, and absolute ethanol, according to conventional IHC protocols.

C.3.2.3 LA-ICP-MS of Breast Cancer Tissue Sections The tissue sections, mounted on Superfrost Plus slides, were inserted into the two volume cell and analyzed by LA-ICP-MS (Element XR coupled to New Wave 213). The two volume cell offers a 100 mm × 100 mm × 25 mm large area (L × W× H) for sample positioning, which is depicted in Fig. C.3.2.3-1. For laser ablation, a second cell is placed above the large cell. The second cell is constructed as a cylinder, which is approximately 20 mm in diameter, and a height of approximately 10 mm. It is positioned above the ablation site, from where the aerosol is transported to the ICP, and the large cell is moved below by a xy-stage during ablation of the sample. The second cell is displayed in Fig. C.3.2.3-2. The operating conditions of the LA-ICP-MS measurements are given in Table C.3.2.3-1. Since the New Wave 213 is equipped with a beam expander, the laser spot size is adjustable between 4 and 250 µm. The isotopes monitored were 63Cu, 65Cu, 66Zn, 153Eu, 159Tb, 165Ho, and 169Tm. However, to achieve highest sensitivity for lanthanides during transient signals, Cu and Zn isotopes were removed from the ICP method for multiplex experiments to avoid a further magnet settling time.

52 BAM-Dissertationsreihe Part C: Experimental

Figure C.3.2.3-1 100 mm × 100 mm large area for sample positioning in the ‘two volume cell’ by New Wave.

Figure C.3.2.3-2 Second cell of the ‘two volume cell’ by New Wave. Figure courtesy of New Wave.

53 Part C: Experimental

Table C.3.2.3-1 Operating conditions of LA-ICP-MS for IHC analyses.

Laser ablation

Laser New Wave 213

Ablation chamber Two volume cell

Laser energy [%] 35

-2 Fluence [J cm ] 200 µm spot: ~ 0.1–0.3 J cm-2 (beam expander) Imaged aperture:~ 0.5–0.8 J cm-2 (< 110 µm)

Laser spot size [µm] 25–200

Scan rate [µm s-1] 25–200

Repetition rate [Hz] 10–20

Ablation mode Adjacent single line scans

ICP-MS

Instrument Element XR

Sampler and skimmer cone Nickel

RF power [W] 1350

Carrier gas, He [L min-1] 1

Nebulizer gas, Ar [L min-1] 0.8

Auxiliary gas, Ar [L min-1] 0.8

Plasma gas, Ar [L min-1] 15

Data acquisition

Isotopes monitored (1) 153Eu, 159Tb, 165Ho, 169Tm

(2) 63Cu, 65Cu, 66Zn, 153Eu, 159Tb, 165Ho, 169Tm

Samples per Peak 100

Mass Window [%] 5

Sample integration time [ms] 2

Total integration time [ms] 10

Total time per line scan Typically 1 min, dependent on the scan rate, and the size of the tissue.

Total analysis time Typically 1 h for breast cancer tissue at 200 µm laser spot size, and 200 µm s-1 scan rate.

54 BAM-Dissertationsreihe Part C: Experimental

C.4 Labeling of Single Cells and Tissues by Iodination Iodination has been used to stain protein spots for analysis by LA-ICP-MS of Western blot membranes.60 In this work, it was applied for the first time to the labeling of single cells and tissue sections and hence, was optimized accordingly. Furthermore, iodine was tested as an internal standard for thin sections during LA-ICP-MS-based IHC analyses to correct for tissue inhomogeneities.

C.4.1 Iodination of Thin Sections Solutions were prepared using Milli-Q water. Iodine (Applichem, Darmstadt, Germany) was added until saturation to 50 mM KI (Applichem, Darmstadt, Germany) while stirring. An aliquot of 100 µL KI3 solution was placed onto a 5 µm tissue section for 30, 60, and 90 seconds. The reaction was stopped by reducing elemental iodine to iodide with 100 µL 50 mM Na2S2O4 (Merck, Darmstadt, Germany) for 3 minutes. Rinsing was conducted with PBS-T (phosphate buffered saline pH 7.3, 137 mM

NaCl, 2.7 mM KCl, 4.3 mM Na2HPO4, KH2PO4 (all from Merck, Darmstadt, Germany) and 0.05 % (v/v) Tween 20 (Applichem, Darmstadt, Germany)).

C.4.2 Fixation and Iodination of Fibroblast Cells For optimized LA-ICP-MS, fixation and dehydration of iodinated cells are important to retain cell morphology during laser ablation. Swiss albino mouse fibroblast cells (cell line 3T3; DSMZ, Braunschweig, Germany) were grown as monolayer on sterile coverslips (Thermo Fisher Scientific, Waltham, USA) for 48 hours in Dulbecco’s modified eagle medium supplemented with 10 % fetal calf serum and 1 % ZellShieldTM (all from Biochrom AG, Berlin, Germany) in a humidified environment

(37°C and 5 % CO2). For the iodination procedure, 3T3 cells were washed thoroughly with PBS (Biochrom AG, Berlin, Germany), iodinated, and fixed with 4 % para- formaldehyde in PBS for 15 min.

A slight variation of the iodination protocol was applied for fibroblast cells: KI3 was diluted 1:10 in PBS (Biochrom AG, Berlin, Germany), and the reaction was stopped with 50 mM Na2S2O4 in PBS for 60 s, followed by washing the cells in PBS. During iodination of fibroblast cells, a deep brown staining of cell nuclei was observed, which started to spread out to the cytoplasm after 60 s.

55 Part C: Experimental

The brown color disappeared as soon as the reaction was stopped by sodium dithionite. As last step, the cells were dehydrated in a graded series of ethanol.

C.4.3 LA-ICP-MS of Iodinated Cells and Tissue Sections For LA-ICP-MS of single cells and tissue sections, the operating conditions of the LA-ICP-MS measurements were individually optimized for each sample. These include laser energy, spot size, scan rate, repetition rate, and gas flows. Typical settings are given in Table C.4.3-1.

56 BAM-Dissertationsreihe Part C: Experimental

Table C.4.3-1 Operating conditions of LA-ICP-MS for iodinated tissues and cells.

Laser ablation

Laser New Wave 213, two volume cell

Ablation chamber Two volume cell

Laser energy [%] 35

Energy denstiy [J cm-2] 200 µm spot: ~ 0.1–0.3 J cm-2 (beam expander) Imaged aperture:~ 0.5–0.8 J cm-2 (< 110 µm)

Laser spot size [µm] 4–200

Scan rate [µm s-1] 5–200

Repetition rate [Hz] 5–20

Ablation mode Adjacent single line scans

ICP-MS

Instrument Element XR

Sampler and skimmer cone Nickel

RF power [W] 1350

Carrier gas, He [L min-1] 1

Nebulizer gas, Ar [L min-1] 0.8

Auxiliary gas, Ar [L min-1] 0.8

Plasma gas, Ar [L min-1] 15

Data acquisition

Isotopes monitored 127I, 153Eu, 159Tb, 165Ho, 169Tm

Samples per Peak 100

Mass Window [%] 5

Sample integration time [ms] 2

Total integration time [ms] 10

Total time per line scan 1–3 min, dependent on the scan rate, and the size of the tissue.

Total analysis time 15 min–4 h, dependent on the sample analyzed.

57 Part C: Experimental

C.4.4 Thyroid Gland Natural iodine is stored in thyroid glands, and was analyzed by LA-ICP-MS to monitor the distribution of iodine in individual glands. For this purpose, formalin-fixed, paraffin-embedded human thyroid gland was sectioned at a thickness setting of 3 µm by a microtome. Thin sections were dewaxed (refer to section C.3.2.1) prior to laser ablation, which was performed according to section C.4.3.

C.5 Gel Electrophoresis Gel electrophoresis was conducted at Proteome Factory AG, Berlin in collaboration with Estefanía Moreno-Gordaliza and Karola Lehmann.

C.5.1 Chemicals Ethanol and acetic acid were obtained from Carl Roth (Karlsruhe, Germany). Hydrochloric acid was obtained from Merck (Darmstadt, Germany). All solutions were prepared with Milli-Q water. All other reagents were obtained from Sigma Aldrich (Taufkirchen, Germany), unless otherwise stated.

C.5.2 Incubation of Standard Proteins with Cisplatin The interaction of cisplatin with proteins is not yet fully understood and hence, the interaction of cisplatin with five standard proteins was investigated here. Human serum transferrin (TF), human serum albumin (HSA), carbonic anhydrase from bovine erythrocytes (CA), horse heart myoglobin (MYO), and equine cytochrome c (CYT C)) at a concentration of 60 µM were incubated separately with cisplatin (Sigma-Aldrich) at a molar ratio 1:10 (protein:drug) in a buffer containing NaCl

(4.64 mM) and Tris-NO3 (10 mM, pH 7.4), reproducing the physiological intracellular saline and pH conditions, at 37 °C for 96 h. Unreacted cisplatin was removed from the solutions with 3 kDa spin-cut-off filters (Amicon Ultra -0.5 mL Ultracel-3, Millipore, USA) by centrifugation (Eppendorf 5415R, Eppendorf, Germany) at 14000 g for 30 min, followed by a further washing step of the retained protein fraction with the incubation buffer.

58 BAM-Dissertationsreihe Part C: Experimental

C.5.3 Cell Cultures and Protein Extraction As a model system for the kidney, renal proximal tubule epithelial cells (RPTECs) from pig kidney were obtained as described by Pérez et al.,154 and were incubated with 1 mM cisplatin (Pharmacia, Barcelona, Spain) for 6 hours. Cells were scraped and lysed in 400 µL of lysis buffer at 70 ºC (2 % (w/v) SDS; 19 % (v/v) glycerol; 790 mM Tris HCl pH 6.8 in Mili-Q water, 1 µL mL-1 phenylmethylsulfonyl fluoride (PMSF), and 10 µL mL-1 protease inhibitors). Cell lysates were heated at 100 ºC for 5 minutes, homogenized on ice and centrifuged at 12 000 g for 5 minutes at 4 ºC. Supernatant containing cytosolic proteins extract was separated. Proteins were precipitated by adding ten volumes of acetone at -20 ºC to a volume of 150 µL of extract, and by incubation for 30 min at -20ºC, followed by centrifugation at 13 000 g at 4 ºC for 30 min. The supernatant was removed and discarded, and the protein pellet was washed three times with 100 µL 80 % acetone, each washing followed by centrifugation at 13 000 g at 4 ºC for 3 min and removal of supernatant. Finally the protein pellet was resuspended in a buffer containing 8 M Urea and 10 mM Tris-HCl pH 7.4. Total protein content in the protein extract was determined with a commercial microplate protein assay (Bradford, BioRad).

C.5.4 One Dimensional Electrophoresis (SDS-PAGE) Pt-bound standard proteins were diluted appropriately and mixed 1:1 with Lämmli sample buffer (2 × LSB). When reducing conditions were applied, 5 % 2-mercaptoethanol was added to the 2 × LSB. The mixtures were heated for 5 min at 95 ºC for the denaturation of proteins.

Table C.5.4-1 Buffers used for 1D-SDS-PAGE.

Buffer Composition Lämmli sample buffer (2×) 125 mM Tris-HCl pH 6.8 6 % glycerol 2 % SDS 0.025 % bromophenol blue (BioRad) Running buffer 25 mM Tris-HCl pH 8.3 200 mM glycine 0.1 % SDS

59 Part C: Experimental

The SDS-PAGE protein separations were performed in a Mini Protean Electrophoresis System (BioRad) using 15-wells, 1-mm-gels with 4 and 12.5 % of polyacrylamide in the stacking and resolving gels, respectively. The composition of the gels is specified in table C.5.4-2. The resolving gel was polymerized for 30 min, followed by the polymerization of the stacking gel for another 30 min. The gel was covered by isopropanol to seal the gel from oxygen. An amount of 500 ng of each protein, either separately or mixed, was loaded on the gels, which were run at 150 V for 75 min at RT in running buffer. Roti-Mark prestained protein standards (Carl Roth GmbH) were also loaded on the gels as molecular weight markers.

Table C.5.4-2 Composition of the stacking (4 %) and of the resolving (12.5 %) gel, volumes are given for one gel. TEMED: Tetramethylethylenediamine, APS: Ammonium persulfate.

Gel Composition Resolving (12.5 %) 2.093 mL Milli-Q water 1.566 mL 40 % Acrylamide:Bisacrylamide 29:1 (BioRad) 1.25 mL 1.5 M Tris-HCl pH 8.5 (BioRad) 50 µL 10 % SDS 2.5 µL TEMED (BioRad) 50 µL 10 % APS

Stacking (4 %) 1.132 mL Milli-Q water 149 µL 40 % Acrylamide:Bisacrylamide 29:1 190 µL 0.5 M Tris-HCl pH 6.8 15 µL 10 % SDS 2.5 µL TEMED 10 % APS

C.5.5 Two Dimensional Electrophoresis (2-DE IEF + SDS-PAGE) For 2-DE separation, the mixture of five Pt-standard proteins was diluted appropriately and was prepared in a final concentration of 8 M Urea. A volume of 2 % of carrier ampholytes (pH 2–4) was added to both the cytosolic protein extract and

60 BAM-Dissertationsreihe Part C: Experimental the standard proteins. Either 2.5 µg of the mixture of standard proteins (500 ng of each protein) or a total amount of 50 µg of the cell culture cytosolic protein extract were loaded in 6 cm IEF gels contained in glass rods for 2-DE separation (Proteome Factory, Berlin, Germany), following a method adapted from that described by Klose and Kobalz.155 Briefly, the separating gel contains 9 M urea, 4 % acrylamide, 0.3 % piperazine diacrylamide (PDA), 5 % glycerol, 0.06 % TEMED, 0.02 % APS, and 2 % ampholytes (pH 3–11). Solutions used during IEF are specified in table C.5.5-1.

Table C.5.5-1 Solutions applied for isoelectric focusing.

Solution Composition Protection solution 5 M urea 5 % glycerol, 2 % ampholytes (pH 2–4)

Cathode solution 9 M urea 5 % glycerol 5 % ethylenediamine

Anode solution 3 M urea 5 % phosphoric acid

Equilibration buffer 3 % SDS 125 mM Tris-HCl pH 6.8 40 % glycerol

A 4 % Sephadex (GE Healthcare) suspension was placed on top of the gel and then proteins were loaded in the anodic side of the gel rod, followed by the protection solution. Isoelectric focusing was performed by applying the cathode and the anode solution in a home-made cell, following a series of running steps: (i) 100 V, for 75 min; (ii) 200 V for 75 min, (iii) 400 V for 75 min; (iv) 600 V for 75 min; (v) 800 V for 10 min and (vi) 1000 V for 5 min. Current and power were set to a maximun of 5 mA and 5 W, respectively and separation was performed at 20 ºC. After separation, gels were equilibrated twice for 5 min in a buffer containing 3 % SDS, 125 mM Tris-HCl (pH 6.8), and 40 % glycerol.

61 Part C: Experimental

For the SDS-PAGE second dimension, the IEF gel grooves were placed onto 1 mm 12.5 % polyacrylamide gels, followed by a 200 µL layer of 1 % agarose

(BioRad), 125 mM Tris-H3PO4 (pH 6.8), 0.1 % SDS, and 0.01 % bromophenol blue. Gels were run at 150 V for 75 min at RT.

C.5.6 Protein Fixation and Staining After electrophoretic separation, proteins were fixed on the gel with a solution containing 50 % ethanol and 10 % acetic acid for 30 min. For protein visualization, gels were stained with Coomassie Brilliant Blue G-250 (BioRad) (2 g L-1 CBB dissolved in 50 % ethanol, 10 % glacial acetic acid) overnight, and finally washed with Milli-Q water for 45 min. Alternatively, for silver staining, a FireSilver staining kit (Proteome Factory, Berlin, Germany) was employed. A shaker was used during fixation and staining.

C.5.7 Gel Drying For gel drying prior to LA-ICP-MS analysis, gels were treated with glycerol for 2 min. Next, they were mounted onto Menzel microscope slides (Thermo Scientific, Germany) and heated in an oven (Thermo Scientific, Germany) at 75 ºC for 2 h.

C.5.8 LA-ICP-MS of Dried Gels LA-ICP-MS measurements were performed with the Element XR coupled to the LSX-213 (CETAC). Gels were placed in a laser ablation chamber designed at ETH Zürich, which is depicted in Figure C.7.7-1. The ablation chamber is suitable for large samples, which can be positioned below the ablation window by a manually movable sled. The dimensions of the sled are 120 × 30 mm (L × W), and the whole chamber is 250 mm long. The ablation chamber has a diameter of 46 mm and therefore, provides the maximum length of a single line scan. There is a single He gas inlet, in contrast to the ablation chamber employed in section C.2 for microarray analysis which provided two He gas inlets, and a gas outlet, which is connected to the ablation window. However, large gels were cut into sections since they did not fit as a whole into the laser ablation chamber. The operating conditions of the LA-ICP-MS measurements are given in Table C.5.8-1.

62 BAM-Dissertationsreihe Part C: Experimental

Figure C.5.8-1 Laser ablation chamber designed at ETH Zürich, which was employed for gel analysis. This ablation chamber is suitable for large samples. There is one He gas inlet on the right hand side, and a gas outlet on the center of the ablation window.

63 Part C: Experimental

Table C.5.8-1 Operating conditions of LA-ICP-MS for dried gels. Laser ablation

Laser LSX-213

Ablation chamber Design ETH Zürich

Laser energy [%] 100

Fluence [J cm-2] 7.3

Laser spot size [µm] 200

Scan rate [µm s-1] 30–100

Repetition rate [Hz] 20

Ablation mode Single line scans

ICP-MS

Instrument Element XR

Sampler and skimmer cone Nickel

RF power [W] 1350

Carrier gas, He [L min-1] 0.9

Nebulizer gas, Ar [L min-1] 0.8

Auxiliary gas, Ar [L min-1] 0.8

Plasma gas, Ar [L min-1] 15

Data acquisition

194 195 196 Isotopes monitored Pt, Pt, Pt Samples per Peak 100

Mass Window [%] 5

Sample integration time [ms] 2

Total integration time [ms] 10

Total time per line scan 3–20 min, dependent on the scan rate, and the length of the monitored gel fraction.

Total analysis time 3 min–3 days, dependent on the size of the sample analyzed (single protein spots or whole 2D gels).

64 BAM-Dissertationsreihe Part C: Experimental

C.6 Study of Renal Protection in Rats Treated with Cisplatin

C.6.1 Drugs Cisplatin was obtained from Pharmacia (Barcelona, Spain). Crystalline cilastatin was kindly provided by Merck Sharp and Dohme S.A., (Madrid, Spain). Both drugs were dissolved in saline solution (0.9% NaCl, Braun Medical S.A, Barcelona, Spain) and administered to the rats intraperitoneally (i.p.).

C.6.2 Rat Kidney Sample Preparation Treatments were applied on seven week old, female Wistar rats (WKY) (Criffa, Barcelona, Spain). The animals were treated at the Hospital General Universitario Gregorio Marañón (Madrid, Spain). Rats were housed in controlled light (12 h light / dark cycle), temperature and humidity conditions with free access to food and water. An acclimatization period of seven days was allowed to the rats before commencing experiments. Animals were handled following the guidelines of the National Council for Care of Laboratory Animals.

Rats were divided into groups (n = 4 animals) according to the treatments received: untreated control rats; cisplatin-injected rats, and cilastatin-treated cisplatin- injected rats. Saline solution (control rats) was administered by a single injection to rats in the same manner and volume (10 mL kg-1 body weight, bw) as cisplatin (5 mg kg-1 bw). Cilastatin was injected every 12 hours from the start of the experiment until the day of the sacrifice at 75 mg kg-1 bw. The first dose of cilastatin was injected just before the administration of cisplatin. Cilastatin was replaced by 0.9 % saline (0.25 mL, 100 g-1 bw) for the untreated and cisplatin-injected rats. Rats were sacrificed on the 5th day after the first injection. For comparison, another rat was treated with a single injection of 16 mg kg-1 bw cisplatin, and was sacrificed on the 3rd day.

Animals were anesthetized, sacrificed, and blood was extracted from the abdominal aorta. After blood extraction, flow was stopped in the rats kidneys by aorta clamping. For elemental imaging, complete perfusion of the kidneys is required. Remaining blood can produce misleading results on elemental distributions in the organ. Thus, the inferior vena cava was punctured and kidneys were perfused

65 Part C: Experimental through a cannula with ice cold saline solution. Kidneys were removed, decapsulated, and immediately placed in 5 % formaldehyde, where they were kept at RT for 48 hours.

After formalin fixation, tissues were dehydrated by a series of ethanol solutions at RT: (70 % ethanol, 30 min; 90 % ethanol, 2 × 30 min; ethanol, absolute, 2 × 30 min). Further treatment in xylene for 1h was repeated twice, prior to paraffin-embedding. FFPE rat kidney tissue was sectioned sagittally at 3 µm thickness setting with the SM 2010R microtome at HELIOS Klinikum Emil von Behring, and mounted onto Superfrost Plus slides. For tissue dewaxing, the sections were treated with xylene, and dehydrated by a series of graded alcohols prior to laser ablation.

C.6.3 HE Staining Hematoxylin and eosin (HE) staining was conducted at HELIOS Klinikum Emil von Behring using standard protocols.

Formalin-fixed, paraffin-embedded tissues were sectioned with a thickness setting of 3 µm for HE staining. Sections were mounted onto Superfrost Plus slides and dewaxed with xylene (Baker) followed by a series of graded alcohols (Thermo Fisher Scientific, Germany). Thin sections were processed by the fully automated HMS 760 Robot Stainer (Microm, Thermo Fisher Scientific, Walldorf, Germany), following the protocol specified in Table C.6.3-1. Hematoxylin II was purchased from Merck (lot HX094620). Shandon Eosin Y, alcoholic, (lot 184317) was from Thermo Fisher Scientific, Germany. The HCl solution specified in table C.6.3-1 was obtained from Hollborn und Söhne, Leipzig, Germany. As a last step, sections were coverslipped.

66 BAM-Dissertationsreihe Part C: Experimental

Table C.6.3-1 Protocol for HE staining with the HMS 760 Robot Stainer.

Solution Rinsing duration Xylene 2 × 4 min Ethanol, absolute 30 s Ethanol, 96 % 90 s Ethanol, 70 % 90 s Water, distilled 90 s Hematoxylin II 6 min Tap water 60 s HCl, 1 % in alcohol, absolute 2 s Tap water 5 min Eosin Y, alcoholic 20 s Ethanol, 70 % 20 s Ethanol, 96 % 60 s Ethanol, absolute 2 × 60 s Xylene 2 × 60 s

C.6.4 LA-ICP-MS of Rat Kidney Tissue The rat kidney tissues were analyzed similar to the breast cancer tissues (section C.3.2.3). The operating conditions of the LA-ICP-MS measurements are given in Table C.6.4-1.

67 Part C: Experimental

Table C.6.4-1 Operating conditions of LA-ICP-MS for rat kidney tissue analysis.

Laser ablation

Laser New Wave 213

Ablation chamber Two volume cell

Laser energy [%] 33

Fluence [J cm-2] 200 µm spot: ~ 0.1–0.3 J cm-2 (beam expander) Imaged aperture:~ 0.5–0.8 J cm-2 (< 110 µm)

Laser spot size [µm] 8–100

Scan rate [µm s-1] 10–150

Repetition rate [Hz] 20

Ablation mode Adjacent single line scans

ICP-MS

Instrument Element XR

Sampler and skimmer cone Nickel

RF power [W] 1350

Carrier gas, He [L min-1] 1

Nebulizer gas, Ar [L min-1] 0.8

Auxiliary gas, Ar [L min-1] 0.8

Plasma gas, Ar [L min-1] 15

Data acquisition

63 65 66 194 195 196 Isotopes monitored Cu, Cu, Zn, Pt, Pt, Pt Samples per Peak 100

Mass Window [%] 5

Sample integration time [ms] 2

Total integration time [ms] 10

Total time per line scan Typically 1–1.5 min, dependent on the scan rate,and the size of the tissue.

Total analysis time Typically 3 h for a whole rat kidney tissue at 100 µm laser spot size, and 150 µm s-1 scan rate.

68 BAM-Dissertationsreihe Part D: Results and Discussion

Part D: Results and Discussion Bioanalytical procedures, making use of organic dyes for detection, often lack standardization. This especially holds true for immunohistochemical analyses, albeit those methods are urgently needed to obtain reliable and reproducible data.12 On the contrary, the detection by ICP-MS is independent of structure and hence, facilitates a standardized and reproducible external calibration. Therefore, this technique is a promising tool for bioanalytical methods. Several genomics and proteomics techniques have been employed in this work for the sensitive detection of highly diverse biomolecules. Since most of these applications had not been used for (LA)-ICP-MS detection before, all parameters involved needed careful optimization.

D.1 ICP-MS-linked Immunoassay for Ochratoxin A Determination in Wine Ochratoxins are mycotoxins produced by two genera of fungi: Aspergillus grows at temperatures in a range from 12–37 °C, and Penicillium in a range from 4–31 °C,156 with ochratoxin A (OTA) as the most prevalent species. These fungi grow in and on a wide range of commodities. Hence, OTA is a common contaminant of different types of foods and beverages, such as wine, beer, coffee, grape juice, meat, dairy products, and spices.157 It was shown that OTA has a high affinity to blood proteins such as serum albumin and globulins,158 which leads to a wide distribution into all organs, with high levels in the liver and in the kidney.159 Moreover, results indicate that it is carcinogenic in rats and mice.160 Consequently, limit values for OTA in a number of foodstuffs have been established in Directive EC 1881/2006161 and 105/2010.162 The maximum level of OTA for wine and grape fruits was set to 2 µg kg-1,162 which documents the need for a reliable analysis of comestible goods. Ochratoxin A determination in wine is mainly conducted by means of high-performance liquid chromatography and mass spectrometry (HPLC-MS). This method is cumbersome since OTA has to be extracted from wine samples followed by a clean-up procedure.163 Furthermore, stable isotope labeled internal OTA standards have to be synthesized for accurate quantification.164 By means of LC-MS, detection limits for OTA in wine of 0.002 up to 0.01 µg L-1 were reported,165 and are similar to other matrices like milk (LOD: 0.002 µg L-1).166 An advantage of this method

69 Part D: Results and Discussion is the ability to separate OTA and other ochratoxin species such as OTB,167 which up to now cannot be distinguished by the available monoclonal antibodies against OTA.

Immunoassays, on the other hand, are well established for the fast and reliable detection of antigens and haptens in a variety of biological matrices with no need for sample clean-up. This technique is a powerful tool for many diverse applications such as HIV testing,168 vaccine studies,169 or the quantification of caffeine in surface waters.170 Hence, an immunoassay for OTA detection was developed at BAM with aqueous OTA standards, a LOD of 0.01 µg L-1, and a limit of quantification (LOQ) of 0.037 µg L-1.171 Consequently, it was needed to improve both LOD and LOQ to meet the standards set by LC-MS, and to investigate the application of this assay to OTA determination in a real matrix, e.g. in wine.

As has already been discussed in part B, the sensitivity of ICP-MS is proportional to the number of labels conjugated and hence, the application of nanoparticles is advantageous for signal amplification. Müller et al. employed antibodies conjugated with 10 nm gold nanoparticles, and calculated a mean of 53 000 gold atoms per antibody, which is consistent with one 10 nm nanoparticle.56 Here, anti-mouse IgG antibodies were conjugated with 40 nm gold nanoparticles, according to the manufacturer. Consequently, not more than one 40 nm gold nanoparticle should be attached per antibody, containing approximately 3.5 × 106 gold atoms, as estimated from their volume. It was of interest to examine the influence of ICP-MS as a detection tool for an immunoassay, since the LOD for horseradish peroxidase with

3,3’,5,5’-Tetramethylbenzidine (TMB) / H2O2 as substrate (photometry) was determined as 2 × 10 15 mol L-1 by Porstmann,172 which is equivalent to 1 × 109 labels L-1. On the contrary, the application of gold nanoparticles as elemental labels to ICP-MS should provide a LOD of 2 × 106 labels L-1, taking into account that one 40 nm nanoparticle contains about 3.5 × 106 gold atoms, and a LOD for gold of 2 ng L-1.

For comparison of both detection methods, an immunoassay for OTA determination in wine was elaborated and will be introduced in the following sections. Labeled secondary antibodies were applied for detection, conjugated with horseradish peroxidase and with gold nanoparticles, respectively, as has already been mentioned in the experimental section. A typical standard curve of a

70 BAM-Dissertationsreihe Part D: Results and Discussion competitive immunoassay is presented in Fig. D.1-1, following a sigmoidal distribution. Thus, high analyte concentrations result in a weak signal, and vice versa.

Figure D.1-1 Sigmoidal response based on the 4-parameter log-fit for an indirect competitive immunoassay.

D.1.1 Minimization of Nonspecific Binding In an immunoassay experiment it is essential to minimize nonspecific binding in order to improve the signal-to-background ratio and thus, the limit of detection. It is very common to add BSA as a blocking agent during incubation of the primary and secondary antibody for minimization of nonspecific binding. However, this approach was not feasible for the immunoassay presented herein. In the presence of BSA in the incubation steps of the assay, the calibration curve (see Fig. D.1.1-1) exhibits an increased signal at 100 µg L-1 OTA, and already reaches a plateau at 10 µg L-1 OTA. This observation leads to the assumption that free OTA is mainly bound to BSA during incubation. It is reported in the literature that OTA accumulates in plasma,159 and the findings made herein confirm a strong binding of OTA to serum albumin. The anti-OTA antibody mainly binds immobilized OTA-BSA conjugate, due to a lack of free OTA in solution. Thus, the detection signal increases already at high OTA concentrations, preventing a sensitive detection. On the other hand, the use of 0.1 % casein as an additive in all antibody solutions could consequently minimize nonspecific binding (see Fig. D.1.1-2), and a LOD of 0.003 µg L-1 was achieved for OTA determination.

71 Part D: Results and Discussion

1.0

0.8

0.6

0.4 normalized intensities (197Au / (191Ir + 193Ir)) 0.2

0.0 1E-801E-5 1E-4 1E-3 0.01 0.1 1 10 100 OTA [µg/L]

Figure D.1.1-1 Optimization of OTA immunoassay standard curve. Standards in Milli-Q water, antibodies in PBS (0.1 % BSA); Detection of digested gold labels by ICP-MS. Figure adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

72 BAM-Dissertationsreihe Part D: Results and Discussion

1.2

1.0

0.8

0.6

0.4 normalized O.D.

0.2

0.0 1E-80 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 1000 OTA [µg/L]

Figure D.1.1-2 Optimized OTA immunoassay standard curve with 0.1 % casein, photometric detection. OTA standards were diluted in 100 mM Tris (0.1 % casein), pH 7.5. Fit parameters: A = 0.97, D = 0.02, C = 0.04, B = 1.38, R² = 0.99671. Figure adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

D.1.2 Immunoassay Digestion for ICP-MS Detection The digestion kinetics of gold nanoparticle labeled anti-mouse IgG antibodies on the well plates was studied. For this purpose, the optimized assay – employing 0.1 % casein – was performed with a 0.0001 µg L-1 OTA standard. Dissolution with 5 % (v/v) digestion acid was conducted for 190 minutes, and aliquots were taken after 10, 20, 30, 60, 180, and 190 minutes. To ascertain that all gold nanoparticles were dissolved, a similar sample was dissolved with 20 % (v/v) digestion acid, and an aliquot was taken after 300 minutes. No increase in gold signal was observed, supporting that digestion was complete and thus, this data point was defined as 100 % recovery. As evident from Fig. D.1.2-1, 45 % of the gold nanoparticles had already been digested after ten minutes. Subsequent dissolutions were conducted for

73 Part D: Results and Discussion five hours with 20 % (v/v) digestion acid to assure a complete dissolution of the gold nanoparticles.

Figure D.1.2-1 Digestion kinetics of the gold nanoparticle label monitored by ICP-MS. Up to three hours digestion in 5 % (v/v) digestion acid (triangle), 5 hours digestion in 20 % (v/v) digestion acid (square) for comparison. Figure adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

D.1.3 Optimization of Wine Sample Detection The determination of OTA in aqueous standards was achieved with a LOD of 0.003 µg L-1. As limit values for OTA exist in wine, it is of great interest to perform the developed immunoassay in a biological matrix.

For the determination of OTA in a red wine reference material with an OTA content of 0.60 ± 0.14 µg L-1, the wine was diluted 1:20 to minimize matrix interference. However, dilution in Milli-Q water was ineffective and OTA content was highly overestimated. In a study with photometric detection, OTA standards and wine samples were diluted in Milli-Q water, compared to the addition of 0.1 % casein, 0.1 % PEG, or 0.1 % gelatin. Results are displayed in Table D.1.3-1. Most likely

74 BAM-Dissertationsreihe Part D: Results and Discussion polyphenols in wine interact with the antibody and as a consequence, OTA can no longer bind to the antibody neither in solution nor immobilized on the well plate. The results indicate that matrix suppression was most efficient for application of casein. Interestingly, gelatin and casein are known to be useful additives in wine processing.174 However, even with the addition of these substances to sample and standards OTA content in the red wine reference material was overestimated in all samples.

Table D.1.3-1 Optimization of photometric detection. Comparison of different additives for wine dilution. OTA content of red wine reference material: 0.60 ± 0.14 µg L -1. Uncertainty is given as ± standard deviation (n = 4). Table adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

Milli-Q Milli-Q Milli-Q Milli-Q water 100 mM Tris water / 0.1 % water / 0.1 % water / 0.1 % pH 7.5 / 0.1 % casein PEG gelatin casein

OTA [µg L-1] 2.95 ± 0.08 5.72 ± 1.22 2.52 ± 0.28 14.44 ± 1.35 1.13 ± 0.05

Additives for wine dilution were also tested during ICP-MS detection. It is evident from Fig. D.1.3-1 that gelatin is an unfavourable additive in this case, as it results in very low measurement precision and high background, which might be due to nonspecific binding of gold ions to the gelatin network. Contrarily, by diluting the wine in 100 mM Tris pH 7.5 / 0.1 % casein, accuracy and precision was highly improved to 1.13 ± 0.05 µg L-1 OTA in the red wine reference material. Results for ICP-MS and photometric detection are displayed in Fig. D.1.3-2 and Fig. D.1.1-2, respectively. Consequently, 100 mM Tris pH 7.5 / 0.1 % casein was used for all further OTA determinations in wine.

75 Part D: Results and Discussion

1.2

1.0

0.8

0.6

0.4 197Au / (Ir191 + Ir193)) + (Ir191 / 197Au normalizedintensities ( 0.2

0.0 1E-80 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 1000 OTA [µg/L]

Figure D.1.3-1 Optimization of OTA immunoassay standard curve with gelatin. Standards in Milli-Q water (0.1 % gelatin), detection of the gold nanoparticle label by means of ICP-MS. Fit parameters: A = 0.96, D = 0.20, C = 0.03, D = 0.58, R² = 0.92941. Figure adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

1.2

1.0

0.8

0.6

0.4 normalizedintensities (197Au / (Ir191 + Ir193)) + (Ir191 / (197Au 0.2

0.0 1E-80 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 1000 OTA [µg/L]

Figure D.1.3-2 Optimized OTA immunoassay standard curve with casein. Standards in 100 mM Tris (0.1 % casein), pH 7.5, detection of the gold nanoparticle label by means of ICP-MS. Fit parameters: A = 0.96, D = 0.006, C = 0.04, D = 0.89, R² = 0.99284. Figure adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

76 BAM-Dissertationsreihe Part D: Results and Discussion

D.1.4 Figures of Merit for Photometry and ICP-MS Detection The LOD of the assay was determined at 85 % signal intensity, which corresponds to the 3s definition assuming a 5 % relative standard deviation.142 For ICP-MS and photometric detection, the LOD for OTA was 0.003 µg L-1, if 0.1 % casein was applied to all antibody solutions. Due to the sigmoidal calibration function, error distribution of the concentration is heteroskedastic. This results in an increase of uncertainty for very high and very low values of the standard curve. The limit of quantification was therefore determined by means of the precision profile developed by Ekins.143 In this work, a relative uncertainty of 40 % was defined as acceptable. A quantification range of 0.01-1 µg L-1 was calculated for ICP-MS and for photometric detection, as illustrated in Fig. D.1.4-1 a and b, respectively.

White and red wine, and a red wine reference material were analyzed for OTA determination. OTA concentrations varied from 0.60 µg L-1 up to 560 µg L-1, and dilution factors from 1:20 to 1:2000. Results are summarized in Table D.1.4-1. The red wine reference material has an OTA content of 0.60 ± 0.14 µg L-1 of natural origin. Appropriate extraction, followed by HPLC separation, was applied prior to fluorescence or MS/MS detection techniques during certification.175 The content of other ochratoxins was not certified.

a 200

180 1.0

160 0.8 140

120 0.6 100

80 0.4 60 relative uncertaintyrelative[%]

40 normalizedintensities 0.2 193Ir)) + (191Ir / (197Au 20

0 0.0 1E-80 1E-5 1E-4 1E-3 0.01 0.1 1 10 100 OTA [µg/L]

Figure D.1.3-1 a OTA immunoassay standard curve with ICP-MS detection and precision profile. Fit parameters: A = 0.94, D = 0.01, C = 0.03, D = 1.14, R² = 0.99943.

77 Part D: Results and Discussion

Figure D.1.3-1 (continued) b OTA immunoassay standard curve with photometric detection and precision profile. Standards and wine samples in 100 mM Tris (0.1 % casein), pH 7.5. Fit parameters: A = 0.93, D = 0.01, C = 0.04, D = 1.19, R² = 0.99993. The limit of quantification at 40 % relative uncertainty is illustrated by a dashed black line. The quantification range for OTA in wine is represented by those concentrations of the precision profile curve (black line with black crosses), which are located below the 40 % line. Red squares, blue circles, and black triangles: Replicates in the immunoassay. Figures adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

78 BAM-Dissertationsreihe Part D: Results and Discussion

Table D.1.4-1 Determination of OTA in wine with ICP-MS and photometric detection. n = 4 if not stated otherwise. Uncertainty is given for 95 % confidence interval; n. d. = not detectable. Sample dilution in 100 mM Tris / 0.1 % casein, pH 7.5. Table adapted from reference 173, courtesy to Journal of Analytical Atomic Spectrometry.

Material ICP-MS Photometry Dilution [µg L-1], [µg L-1], Recovery [%] Recovery [%] Candidate reference material, 0.78 ± 0.12, 1.13 ± 0.05, 1:20 unspiked 0.60 ± 0.14 µg L-1 OTA 130 188 Candidate reference material, 145 ± 15, 143 ± 6, 1:1000 spiked with 110 ± 14 µg L-1 OTA 132 130 Candidate reference material, 280 ± 27, 298 ± 22, 1:1000 spiked with 205 ± 14 µg L-1 OTA 137 145 Candidate reference material, 174 ± 29, 190 ± 26, 1:2000 spiked with 205 ± 14 µg L-1 OTA 85 93 Candidate reference material, 675 ± 16, 636 ± 92, 1:1000 spiked with 556 ± 14 µg L-1 OTA 121 114 Red wine, unspiked n. d. n. d. 1:20 White wine, unspiked n. d. n. d. 1:500 Red wine, 125 ± 13, 105 ± 3, 1:500 spiked with 87 ± 14 µg L -1 OTA 144 121 White wine, 196 ± 29, 173 ± 8, 1:1000 spiked with 194 ± 14 µg L -1 OTA 101 89 White wine, n=6 128 ± 9, 128 ± 7, 1:1000 spiked with 82 ± 14 µg L -1 OTA 156 156 White wine, n=6 97 ± 8, 108 ± 5, 1:1000 spiked with 82 ± 14 µg L -1 OTA, matrix matched standards 118 132

OTA content in the red wine reference material was determined as 0.78 ± 0.12 µg L-1 by means of ICP-MS and as 1.13 ± 0.05 µg L-1 by photometric detection. OTA in this red wine is of natural origin and an overestimation might be caused by other ochratoxin species. Thus, the reference material was spiked with 110 ± 14 µg L-1, 205 ± 14 µg L-1, and 556 ± 14 µg L-1 OTA, and was measured in a 1:1000 dilution to meet the quantification range. Results indicate a recovery of

79 Part D: Results and Discussion

132 %, 137 % and 121 % for ICP-MS, and a recovery of 130 %, 145 % and 114 % for photometric detection. By a further dilution (1:2000) of the wine spiked with 205 ± 14 µg L-1, a recovery of 85 % for ICP-MS and 93 % for photometry was determined. It is likely that the immunoassay is affected by matrix effects, since recoveries improved with decreasing matrix effect of the wines in this case.

The tested red and white wine had a natural level of OTA below the LOD of the assay. Spiking of the red wine with 87 ± 14 µg L-1 OTA, followed by ICP-MS and photometric detection resulted in a recovery of 144 % and 121 %, respectively. The white wine was spiked with 194 ± 14 µg L-1 OTA, and was accurately determined by ICP-MS with 101 % recovery, and 89 % recovery by photometric detection. Spiking with 82 ± 14 µg L-1 OTA resulted in an overestimation. Recovery for both detection methods was 156 %. By adding a 1:1000 dilution of the white wine to OTA standards as well, matrix matched standards were prepared. This approach resulted in improved measurement accuracy in this case. A recovery of 118 % and 132 % was achieved by ICP-MS and photometry, respectively.

D.1.5 Summary An immunoassay for OTA determination in wine was successfully developed. Analyses were accomplished with good recoveries at low concentrations in the range of 0.01–1 µg L-1 OTA. With the developed immunoassay, OTA determination in wine was accomplished in the concentration range of the EU limiting value, and even below. It is of great benefit to detect such low concentrations, and to distinguish between contaminated and innoxious wines, applying Directive EC 1881/2006161 and 105/2010,162 with a method as convenient as an immunoassay, which in contrast to HPLC techniques requires virtually no sample preparation. Furthermore, photometric and ICP-MS detection were compared. It is evident from the data that the inherent precision of a particular detection method is not crucial for assay precision in general. The precision profile of ICP-MS detection is similar to the profile of photometric detection, as obvious from Fig. D.1.4-1. It can be concluded that the assay performance is not significantly influenced by the detection system. Instead, major influences arise from other sources in this case, such as the sample position on the well plate or time dependent procedures like incubation steps. Furthermore, other substances in the wine might affect OTA binding to the antibody, despite its high

80 BAM-Dissertationsreihe Part D: Results and Discussion sensitivity. These effects need to be further investigated for improved assay precision. The implementation of an elemental tag might be more advantageous for a noncompetitive assay format. In this case, the wide linear dynamic range of ICP-MS can be fully exploited, as well as its sensitivity, in accordance to the theoretical LOD of 2 × 106 nanoparticle labels L-1.

D.2 DNA Microarray Detection by LA-ICP-MS Fluorescent probes are commonly employed for DNA microarrays, which are queried by means of a laser scanner. However, absolute quantification is not feasible by this technique since the effectiveness of the fluorescent tag is dependent on the vicinity of the sample, and might be prone to quenching effects, as has been discussed in section B.3. Furthermore, the multiplex capabilities of fluorescence detection are confined due to spectral overlap, and a limited availability of parallel excitation sources in the microarray scanner. Thus, for highly multiplexed assays a technique like ICP-MS is promising, which additionally has the potential to produce quantitative data.

In this chapter, first results on this alternative approach are presented, applying 10 nm gold nanoparticle labels for microarray detection. Under optimum conditions, the nanoparticles should be monodisperse to achieve defined MS signals, which can be correlated to a particle’s size. On the other hand, the detection limit of commercial microarray scanners is 0.1 fluor µm-2,176 which corresponds to approximately 800 oligonucleotide molecules employed in this work in a 100 µm microarray spot. A saturated microarray spot most likely contains so many molecules, that an average signal of polydisperse nanoparticles should be sufficient for quantification.

The method development involved (i) printing of amino-modified target oligonucleotide on the substrate, an epoxy-coated microarray slide, and to achieve uniform microarray spots with minimized ring stain due to undesired drying effects (ii) blocking of the substrate prior to hybridization to minimize nonspecific binding of probe DNA, (iii) effective hybridization with biotinylated DNA probe, (iv) blocking of the substrate prior to labeling to minimize nonspecific binding of the label, (v) labeling with gold nanoparticles via streptavidin-biotin-linkage, and (vi) visualization of microarray spots by silver enhancement for LA-ICP-MS analysis.

81 Part D: Results and Discussion

A scheme illustrating the sample preparation is given in Fig. D.2-1. A careful optimization of all sample processing steps is crucial for high detection efficiency and low background contribution, and is elucidated in the following.

Hybridization of probe DNA Labeling

Biotin

Streptavidin-gold nanoparticle 10 nm

Figure D.2-1 Scheme of microarray sample preparation. The immobilized target oligonucleotide is hybridized with biotinylated probe oligonucleotide, which is subsequently labeled via streptavidin-biotin-linkage.

D.2.1 Optimization of Microarray Preparation The printing of target DNA on the substrate was accomplished by a microarray spotter, as described in section C.2. For a printing buffer it had to be considered that at low pH, the amino group is not fully deprotonated and thus, the reaction with epoxy moieties will be less efficient. On the other hand, the coating made of organofunctional silane might become unstable at basic pH. Thus, a pH of 8 was most desirable for the covalent linkage of oligonucleotides to the microarray surface. During immobilization it is crucial that the sample does not dry on the microarray slide until the reaction is completed in order to prevent undesirable drying effects such as ring stains.177 The most uniform microarray spots were created at 65 % humidity, which can be controlled by the spotter. At lower humidity, inhomogeneous drying of the sample solution was observed. At higher humidity, microarray spots were no longer strictly separated from each other. After printing, remaining epoxy moieties were saturated with blocking buffer.

82 BAM-Dissertationsreihe Part D: Results and Discussion

The efficient hybridization was monitored with Cy3 labeled target oligonucleotides at varying concentrations. Excitation of the Cy3 dye was conducted at 532 nm with a microarray scanner, and the resulting microarray image is displayed in Fig. D.2.1-1. The negative control at 0 µM DNA concentration on the right hand side of the slide was clearly distinguishable from positive controls (0.01, 0.02, 0.05, 0.1, and 0.2 µM DNA). The integrated fluorescence was plotted versus the corresponding probe DNA concentration to attain a linear calibration curve depicted in Fig. D.2.1-1. As a result, hybridization proved to be highly efficient, and a relative quantification of probe DNA was feasible by this method. Thus, the conditions for printing, blocking of the substrate, and hybridization could be applied to biotinylated probe DNA, which had been optimized by means of fluorescence detection,.

The labeling of DNA with gold nanoparticles conjugated with streptavidin was critical owing to the high affinity of gold nanoparticles to glass surfaces. Therefore, the microarray slide needed blocking, and different labeling strategies were tested (refer to section C.2). Finally, the application of 10 mg mL-1 PEG in Milli-Q water and SuperBlock T20 Buffer was efficacious in the reduction of nonspecific binding of gold nanoparticles to the microarray glass surface. Additionally, the microarray surface was incubated with 10 mg mL-1 PEG prior to labeling. This resulted in a negative control indistinguishable from background noise in LA-ICP-MS measurements, as illustrated in Fig. D.2.1-2. It is evident from Fig. D.2.1-2 a that there was no detectable nonspecific binding of gold nanoparticles on the substrate, which proved the successful surface blocking method developed herein. The 137Ba signal from the substrate in Fig. D.2.1-2 b is given for illustration of the single laser pulses, which result in microarray slide ablation.

83 Part D: Results and Discussion

Figure D.2.1-1 In this case, a template made of silicone (Sigma-Aldrich, Germany) was applied for immobilization with 20 µM target DNA, and the sample solution was pipetted in the 12 existing wells; Hybridization with Cy3-labeled oligonucleotide was performed in varying concentrations (blank = hybridization buffer, 0.01, 0.02, 0.05, 0.1, and 0.2 µM probe DNA, n = 2). R² = 0.999, (y = 8.23 × 109x – 3.78 × 107). The inserted image shows the scanned microarray slide and was generated by the GenePix software (version 6.0).

Figure D.2.1-2 Microarray slide, immobilization with 20 µM target DNA, hybridization with hybridization buffer (negative control), labeling with SA-Au 1 : 100; The measurement was conducted with an ELAN 6100 DRC+ coupled to an ArF excimer laser at 193 nm; laser 197 ablation in single pulse mode at 160 µm square spot size; a Au signal (dwell time: 50 ms), 137 b Ba signal (dwell time: 1 ms).

84 BAM-Dissertationsreihe Part D: Results and Discussion

Since the gold labeled microarray spots are not visible, the staining is crucial for laser positioning during LA-ICP-MS analysis. The visualization was achieved by silver enhancement, e.g. the catalytic reduction of silver ions on the gold nanoparticle surface to metallic silver.178, 179 Below 20 minutes, the microarray spots were not visible yet. On the other hand, the whole microarray slide was stained black for an incubation time > 20 minutes. Hence, an incubation time of 20 minutes was crucial for optimum staining results. Silver stained microarray spots are displayed in Fig. D.2.1-3.

Figure D.2.1-3 Photographs of silver stained microarray spots: a 20 µM, b 10 µM, and c 1 µM target DNA. The 160 µm square laser spot is highlighted in red.

D.2.2 Single Pulse LA-ICP-MS The signal stability concerning total transported aerosol, aerosol number density, and aerosol dispersion, which are represented by peak area, height, and full width at half maximum (FWHM),180 respectively were monitored during single pulse ablation of 197Au in a NIST 610 glass reference material, according to the operating conditions specified in section C.2. The results of three independent runs are given in Table D.2.2-1. The RSDs of the peak area varied between 10–20 % for Au in NIST 610, which might be explained by an inhomogeneous distribution of Au at a laser spot size of 160 µm. On the other hand, the experimental uncertainties might stem from the ablation process itself, which is influenced by the laser performance, the ablation cell characteristics, and transport efficiency.

As shown in Table D.2.2-1, RSDs of the aerosol number density, which depends on the washout time and transport of the aerosol from the cell, is in the same range as RSDs for the peak area. Fast washout times are crucial for short transient signals

85 Part D: Results and Discussion since it improves signal-to-background ratios.181 If aerosol number density is increased, a faster detection is needed to obtain a sufficient number of data points for analysis. Nevertheless, this was not the main shortcoming here. More importantly, RSDs for aerosol dispersion vary to a significantly larger extent. This parameter is mainly influenced by non uniform gas flow dynamics within the ablation cell. Indeed, the sled position seems affect the signal, which broadened upon ablation on the edges of the chamber window. The value of 76 % RSD for FWHM at n = 10 might be explained by the low number of replicates, an unfavorable position in the ablation chamber, or the low signal intensities.

Table D.2.2-1 Average and RSDs for peak area (total transported aerosol), peak height (aerosol number density), and full width at half maximum, FWHM (aerosol dispersion) for Au in NIST 610. Three runs with n = 18, n = 18, and n = 10, respectively.

Average, RSD [%] Average, RSD [%] Average, RSD [%], n = 18 n = 18 n = 10 Area 1217 14 1360 20 837 10 Height [cps] 1130 13 1220 24 682 16 FWHM [s] 0.83 29 0.94 28 0.57 76

D.2.3 LA-ICP-MS Analysis of DNA Microarrays The measurements presented herein were conducted under optimized conditions as specified in section C.2, and were applied for gold nanoparticle labeling of DNA microarrays. Two exemplary ablation sequences of 0.05 µM, and 10 µM probe DNA are displayed in Fig. D.2.3-1. In this figure, ion intensities for Au measured at m / z = 197 are shown for each laser pulse. Laser ablation was performed in single pulse mode at 160 µm square spot size, which was sufficient to completely ablate microarray spots (approximately 80 µm in diameter, as illustrated in Fig. D.2.1–3). According to the observation that peaks broadened upon ablation on the edges of the chamber window, the last two signals in Fig. D.2.3-1 b were omitted for analysis.

The results of microarray detection at varying probe DNA concentrations are presented in Table D.2.3-1 and Fig. D.2.3-2. The signal intensities increased from 0.01–0.5 µM probe DNA, and varied between 2000 and 3200 cps from 1–10 µM.

86 BAM-Dissertationsreihe Part D: Results and Discussion

Thus, at higher concentrations from 0.5 µm onwards, the microarray assay might be saturated. The highest signal was observed for 20 µm probe DNA, which also exhibited the most intense silver staining (see Fig. D.2.1-3), and the experimental limit of detection had already been achieved at 0.001 µM. The detected signal intensities vary significantly, which might be the result of incomplete ablation or nonuniformity of microarray spots.

Figure D.2.3-1 Microarray slide, immobilization with 20 µM target DNA, labeling with SA-Au (1:100), hybridization with a 0.05 µM, and with b 10 µM probe DNA; The measurement was conducted on an ELAN 6100 DRC+ coupled to an ArF excimer laser at 193 nm; laser ablation in single pulse mode; 197Au signal (dwell time: 50 ms).

87 Part D: Results and Discussion

Table D.2.3-1 Average and RSDs of peak areas for varying probe DNA concentrations. n = number of microarray spots analyzed; n.d. = not detectable.

# Target DNA [µM] n Average RSD [%]

1 20 58 6430 57

2 10 48 2029 29

3 5 43 2246 34

4 2 45 3218 29

5 1 48 2387 48

6 1 60 3155 35

7 0.5 54 3801 34

8 0.2 69 2798 46

9 0.05 46 1616 55

10 0.01 41 826 104

11 0.001 - n.d. n.d.

10000

8000

6000

4000 197Au [cps] 197Au

2000

0.01 0.1 1 10 target DNA [µM]

Figure D.2.3-2 Analysis of microarray slides at 20 µM probe DNA and varying target DNA concentrations. The slides were subsequently labeled with SA-Au (1:100), and silver stained. The slide at 1 µM target DNA was analyzed in two independent runs.

88 BAM-Dissertationsreihe Part D: Results and Discussion

The observed relative standard deviations (RSDs) of the analyzed microarray slides ranged from 29–57 % (0.01-20 µM probe DNA), with high RSDs for 0.01 µM and 20 µM, whereas RSDs improved for medium concentrations (see Table D.2.3-1). The slide with 1 µM probe DNA was investigated in two independent measurement sequences with n = 48 and n = 60 with a RSD of 48 % and 35 %, respectively. These differences in RSD cannot be explained by Poisson-statistics. On the other hand, the generally high RSDs might only partially stem from the measurement process itself, but mainly from inhomogeneities within the microarray samples. Due to the fact that peak area reflects total transported aerosol,180 and since the area of spots increases proportional to r², small variations in size have a high impact on the integrated signal. A solution to this problem might be the use of an internal standard, which is homogeneously distributed in the microarray spots. Black deposits were visible on the microarray slides after silver enhancement indicating an inhomogeneous distribution of silver. Furthermore, for short signals (as from single pulse ablation), spectral skew might have an influence on signal stability due to the sequential detection in Q-ICP-MS, which might be too slow to properly record short transient signals.181 Hence, for the transient nature of LA-ICP-MS signals (and especially for single pulse ablation) a simultaneous signal detection looks more promising, as provided by TOF-MS182 or by Mattauch-Herzog instruments.183, 184

D.2.4 Summary The measurement by LA-ICP-MS revealed high RSDs in the peak areas, which most likely stem from size variabilities in the microarray spots. These differences have a significant influence on signal intensities since the area of spots increases proportional to r². Furthermore, the uniformity of microarray spots is a prerequisite for quantitative analyses. An improvement might be achieved by contactless printing of target DNA. Only if this challenge is tackled, a modified ablation chamber will be of benefit to this special application. Interestingly, the total transported aerosol was not significantly altered for various ablation chambers as shown by Bleiner and Günther,180 and the use of a different ablation chamber most likely will not affect RSDs in this application. Instead, the signal stability concerning aerosol dispersion might be enhanced by different gas flow dynamics in the ablation chamber.

89 Part D: Results and Discussion

The application of gold nanoparticle labels has been investigated in section D.1 and D.2 for an ICP-MS-based immunoassay and microarray detection by LA-ICP-MS, respectively. On the one hand a dramatic gain in sensitivity was achieved by these labels, but on the other hand nonspecific absorption became more evident compared to other labels. Moreover, only a few elemental labeled biomarkers are commercially available, which reduces the number of applications. For this reason the in-house development of labeling techniques looks promising to increase the number of available biomarkers and beyond that, to enable multi element analyses. This approach was followed in the subsequent sections.

D.3 Combination of Immunohistochemistry with Detection by LA-ICP-MS The main goal in IHC analysis is the verification of preliminary, histomorphological diagnoses by molecular markers, as has been mentioned in sections B and C. Their identification is essential for effective treatment concepts and disease prognosis, and thus reliable and reproducible data are crucial for optimum health care.12 In this work, LA-ICP-MS was employed for IHC, in combination with a labeling technique established by Waentig et al. 57 using p-SCN-Bn-DOTA for labeling of primary antibodies. The use of elemental labeled primary antibodies in combination with LA-ICP-MS for IHC detection offers five advantages:

(i) A possibility for quantification – provided suitable standards are available,

(ii) a simultaneous detection of several tumor markers within one tissue section,

even if they are co-localized,

(iii) a shorter analysis time due to the simultaneous detection,

(iv) comparability of expression levels of different tumor markers, and

(v) the correction of tissue inhomogeneities by a suitable internal standard.

It should be mentioned that up to now metal tagged antibodies have been employed for Western Blot assays only, with limits of detection in the sub pmol range.57 However, antigen concentration in tissue samples is unknown to a large extent, and this fact also applies to the tissue samples investigated here. Thus, it was

90 BAM-Dissertationsreihe Part D: Results and Discussion certainly questionable if the sensitivity of metal tagged antibodies could be transferred to an IHC application.

The optimization of this novel approach and the application for breast cancer tissue will be described in the following sections. Various parameters of the IHC procedure (labeling, incubation, and laser ablation conditions) have a direct impact on the selectivity of the labeled primary antibody, and the sensitivity in LA-ICP-MS, respectively. They determine signal-to-background ratio, and consequently spatial resolution in LA-ICP-MS imaging. Hence, sample preparation steps and LA-ICP-MS parameters of the New Wave 213 were investigated in this work. The optimization strategy was aimed at sufficient signal-to-background ratios with concomitant high spatial resolution, and complete ablation of the tissue in the two volume cell.

D.3.1 Labeling with SCN-DOTA: Optimization of Sample Preparation and LA-ICP-MS Measurements In this chapter the optimization of sample preparation steps and LA-ICP-MS measurements of tissue sections will be presented. The labeling of antibodies was adapted from Waentig et al.57 with slight variations for antibodies obtained as a cell culture supernatant, as already mentioned in section C.3. An average number of two labels per antibody was determined,57 which has been verified for labeled anti-MUC 1 in this work (refer to section F.2).

Sample preparation steps such as tissue thickness, incubation time, and antibody concentration, and LA-ICP-MS parameters such as laser spot size, scan speed, repetition rate, and laser fluence were investigated in a breast cancer tissue, which exhibited high expression levels for Her 2, CK 7, and MUC 1.

D.3.1.1 Optimization of Tissue Thickness and Laser Energy Standard protocols used in conventional IHC were applied as a starting point for the optimization of tissue sample preparation. Generally, an optimum tissue thickness is crucial for assessment of tumor markers in immunohistochemically stained thin tissue sections.91 The same applies to LA-ICP-MS measurements. Furthermore, laser energy, laser spot size and scan speed are the important parameters for laser

91 Part D: Results and Discussion ablation measurements since they determine sensitivity, signal-to-background ratio, spatial resolution, and analysis time.

The formalin-fixation is a reversible process. For target retrieval, tissues were heated to 90 °C for 20 min, and due to adhesive effects of Super Frost microscopic slides they stick to the glass surface. This holds true for 3 µm thin slices employed in routine IHC. For LA-ICP-MS detection, it can be expected that signal intensities increase linearly with tissue thickness. Thus, several tissue thicknesses were tested during IHC procedure and during laser ablation in the range from 3–10 µm.

The upper limit was given by the fact that tissues > 10 µm did no longer stick to the glass surface during target retrieval and the tissue was completely destroyed. Tissues cut at 10 µm stuck partially on the glass surface, but other parts of the tissue started to detach or were already destroyed. From 8 µm down to 3 µm, tissue sections were no longer removed from the glass surface during target retrieval, and LA-ICP-MS detection could be performed. However, the 8 µm tissue was prone to splintering during laser ablation at laser energies above 35 % (fluence ~ 0.1– 0.3 J cm-2 at 200 µm spot employing the beam expander), which caused damage to adjacent lines and inhibited imaging of the tissue. On the other hand, the tissue was not ablated completely at lower energies. Hence, a thickness setting of 8 µm was not suitable for tissue imaging in this case. Furthermore, large particles are generated, being torn out of the tissue during splintering, and most likely did not reach the ICP and did not contribute to a detectable signal or can cause contamination.

In contrast, the 5 µm thin section was ablated without splintering of the tissue. Drying of the tissue with a graded series of alcohols as a last step of sample preparation reduced splintering during laser ablation even further. The results from a single line scan by LA-ICP-MS of a 5 µm, and a < 3 µm thin cut incubated with ~ 1 µg mL-1 Her 2 (Ho) for 3 hours are shown in Fig. D.3.1.1-1 a, b. The latter thin section was cut at a thickness setting of 3 µm, albeit the actual thickness is dependent on density and temperature of the paraffin block during sectioning. In this case, the tissue section was ~ 1–3 µm thin. The LA-ICP-MS was optimized as described in section C. Complete laser ablation of the tissues was performed without splintering for both thin sections at 100 µm laser spot size, 90 µm s-1 scan speed, 5 Hz repetition rate, and 35 % laser energy. For higher laser energies, splintering of

92 BAM-Dissertationsreihe Part D: Results and Discussion the tissue was observed. The figure displays a line scan, monitoring the signal intensity in cps of m / z = 165 (Ho) versus the time in s. Laser ablation in Fig. D.3.1.1-1 a starts shortly before the tissue section at t = 0, and leaves the tissue at t = 40 s. Compared to the ~ 1–3 µm thin cut, signal intensities were approximately five times higher for the 5 µm thin cut. Therefore, all tissue samples investigated here were cut at 5 µm if not mentioned otherwise, and analyzed at 35 % laser energy under the optimized conditions.

-1 Figure D.3.1.1-1 a 5 µm thin cut of breast cancer tissue, incubated with ~ 1 µg mL Her 2 (Ho) for 3 hours. Laser parameters: laser spot size 100 µm, scan speed 90 µm s -1, repetition rate 5 Hz, laser energy 35 %. b < 3 µm thin cut of breast cancer tissue, incubated with ~ 1 µg mL-1 Her 2 (Ho) for 3 hours. Laser parameters: laser spot size 100 µm, scan speed 90 µm s -1, repetition rate 5 Hz, laser energy 35 %.

D.3.1.2 Optimization of Incubation Time and Antibody Concentration It is important to find incubation conditions which generate high signal intensities on the one hand, but minimize nonspecific binding on the other hand. Thus, an optimization of sample preparation and incubation time and antibody concentration was needed. Her 2 (Ho) was incubated at ~ 1 µg mL-1 for one hour in comparison to three hours incubation on a 5 µm breast cancer tissue section. The tissue was ablated at 60 µm laser spot size, 60 µm s-1 scan speed, and 35 % laser energy. In case of the tissue which was incubated with Her 2 (Ho) for one hour, the repetition rate was 4 Hz to prevent splintering, whereas the tissue incubated for three hours was ablated at 10 Hz repetition rate. However, total tissue aerosol was comparable

93 Part D: Results and Discussion since the entire tissue was ablated in the single line scan for both cases. Holmium intensities were approximately three times higher for 3 h incubation compared to 1 h incubation, as shown in Fig. D.3.1.2-1 a, b. In Fig. D.3.1.2-1 b, the variations in signal intensity stem from the tissue sample. Intensities were not enhanced by a further increase of incubation time to 4 h. Thus, an incubation time of 3 hours was selected for all further experiments.

Figure D.3.1.2-1 a, b Holmium intensities measured in a single line scan after incubation -1 with Her 2 (Ho) at ~ 1 µg mL , a 1 h, b 3 h. Laser parameters: laser spot size 60 µm, scan speed 60 µm s -1, laser energy 35 %, and repetition rate 4 Hz (a), 10 Hz (b).

Antibody concentration was varied for 3 h incubation time on a 5 µm breast cancer tissue section. Her 2 (Ho) was applied for incubation at a concentration of ~ 1 µg mL-1 and of ~ 0.1 µg mL-1. Corresponding single line scans are presented in Fig. D.3.1.2-1 b, and D.3.1.2-2, respectively. Incubation at ~ 1 µg mL-1 yielded intensities of approximately 1500 cps at a laser spot size of 60 µm, whereas signal was indistinguishable from background for incubation at a concentration of ~ 0.1 µg mL-1. Hence, the higher concentration was employed further on. Purification steps prior to and after the labeling reaction involve loss of antibody, and therefore incubation with even higher antibody concentration was not feasible in this case. Consequently, a slightly lower concentration and longer incubation times were applied for primary antibodies, as compared to conventional IHC. If higher antibody concentrations were applied, incubation times could be reduced.

94 BAM-Dissertationsreihe Part D: Results and Discussion

To summarize, a thickness setting of 5 µm for thin sections, a laser energy of 35 %, a minimum incubation time of three hours, and a primary antibody concentration of ~ 1 µg mL-1 was used for all further laser ablation experiments on this breast cancer tissue.

Figure D.3.1.2-2 Holmium intensities measured in a single line scan after three hours incubation of Her 2 (Ho) on a 5 µm breast cancer tissue section with ~ 0.1 µg mL -1. Laser parameters: laser spot size 60 µm, scan speed 60 µm s -1, laser energy 35 %, and repetition rate 5 Hz.

D.3.1.3 Optimization of LA-ICP-MS Parameters The He carrier gas flow was optimized at 1 L min -1 for fast aerosol washout times in the two volume cell. The ICP was tuned daily for maximum ion intensities and ThO / Th < 1 % on microscopic glass slides. Furthermore, the LA-ICP-MS parameters were optimized, since they determine sensitivity, signal-to-background ratio, spatial resolution, and analysis time as already mentioned before.

Single line scans were performed on a 5 µm breast cancer tissue section, incubated for 3 h with ~ 1 µg mL-1 MUC 1 (Tb). Laser spot size was varied from 25 µm to 200 µm while keeping the repetition rate at 10 Hz, the laser energy at 35 %, and the ratio of laser spot size to scan speed constant. Terbium intensities are depicted in Fig. D.3.1.3-1 a–d. They were lowest for small laser spot sizes and increased for larger spot sizes. Terbium intensities up to approximately 1000 cps could be detected for laser spot sizes as small as 25 µm and are shown in Fig. D.3.1.3-1 a. On the other hand, signal intensities of approximately 1 × 105 cps

95 Part D: Results and Discussion were detected for 200 µm laser spot size in Fig. D.3.1.3-1 d. Consequently, 200 µm laser spot size was employed for analysis of breast cancer tissues since it provided the highest signal intensity.

Laser ablation with repetition rates of 20 Hz yielded higher intensities than with 10 Hz, as illustrated in Fig. D.3.1.3-1 a, g. Therefore, 20 Hz was applied for an optimized analysis. Scan speed was varied from 100 to 200 µm s-1, with 200 µm laser spot size, 20 Hz repetition rate, and 35 % energy. Examples of single line scans are presented in Fig. D.3.1.3-1 e–g. The signal intensities increased with increasing scan speed since more tissue aerosol was produced within the same time frame. At 200 µm s-1, MUC 1 positive tissue sections were still distinguishable from negative sections (refer to section D.3.3.2) and thus, spatial resolution was acceptable at high scan speed. Consequently, IHC with LA-ICP-MS detection can be performed in a short analysis time of only one hour for a whole breast cancer tissue section.

In summary, the optimized laser ablation parameters were 200 µm laser spot size, 20 Hz repetition rate, and 200 µm s-1 scan speed.

96 BAM-Dissertationsreihe Part D: Results and Discussion

a b

c d

Figure D.3.1.3-1 Optimization of laser ablation parameters on a 5 µm breast cancer tissue -1 section, incubated with ~ 1 µg mL MUC 1 (Tb), CK 7 (Tm), and Her 2 (Ho) for 3 h. -1 a laser spot size 25 µm, scan speed 25 µm s , repetition rate 10 Hz, laser energy 35 %, -1 b laser spot size 50 µm, scan speed 50 µm s , repetition rate 10 Hz, laser energy 35 %, -1 c laser spot size 100 µm, scan speed 100 µm s , repetition rate 10 Hz, laser energy 35 %, -1 d laser spot size 200 µm, scan speed 200 µm s , repetition rate 10 Hz, laser energy 35 %.

97 Part D: Results and Discussion

e f

Figure D.3.1.3-1 (continued) Optimization of laser ablation parameters on a 5 µm breast cancer tissue section, incubated with ~ 1 µg mL -1 MUC 1 (Tb), CK 7 (Tm), and Her 2 (Ho) for 3 h. -1 e laser spot size 200 µm, scan speed 100 µm s , repetition rate 20 Hz, laser energy 35 %, -1 f laser spot size 200 µm, scan speed 150 µm s , repetition rate 20 Hz, laser energy 35 %, -1 g laser spot size 200 µm, scan speed 200 µm s , repetition rate 20 Hz, laser energy 35 %.

D.3.1.4 Selectivity of Labeled Tumor Markers The detection of tumor markers by LA-ICP-MS in human breast cancer tissue sections was studied in comparison to conventional IHC staining. All experiments presented in this section were conducted on the same FFPE breast cancer tissue block, and IHC staining was performed with the BenchMark XT as described in section C.3. An alkaline phosphatase conjugated secondary antibody was used for signal amplification. This enzyme catalyzes the reaction of Fast Red with naphthol, resulting in a red color of positively tested cancer cells.

98 BAM-Dissertationsreihe Part D: Results and Discussion

The incubation on a breast cancer tissue section was always accompanied by a parallel incubation on a palatine tonsil tissue section as a negative control, which means that the biomarker of interest is known to be present at low expression levels. Positive and negative controls were cut at the same thickness, mounted onto the same glass slide, and all sample processing steps were performed identically. Primary antibodies Her 2, CK 7, and MUC 1 applied for LA-ICP-MS were labeled with Ho, Tm, and Tb, respectively via a DOTA linker, as elucidated in section C.3.

For analysis of tumor marker expression levels, a 3 µm thin section was immunostained by conventional IHC to assess MUC 1 distribution in the investigated tissue. The tissue section was stained red as illustrated in Fig. D.3.1.4-1 a, and thus can be evaluated as positive for MUC 1. Thin sections of a palatine tonsil tissue were processed in parallel experiments, and were used as negative controls to ensure high selectivity of all reagents employed for IHC analyses. Tissue sections which are negative for the applied tumor markers appear in blue, as illustrated for MUC 1 negative palatine tonsil in Fig. D.3.1.4-1 b. This is due to counter staining of cell nuclei with hematoxylin, a blue dye which becomes macroscopically visible only if Fast Red dye is absent or generated at low levels. Negative controls for Her 2 and CK 7 produced the same result and are not shown here. Application of Her 2 to a different section resulted in the bright red stain shown in Fig. D.3.1.4-1 c, indicating a pronounced distribution of c-erbB-2 oncoprotein in the breast cancer tissue. Additionally, CK 7 was positively tested on a parallel thin section as depicted in Fig. D.3.1.4-1 d. Moreover, cytokeratin 7 seems to be expressed at lower levels than c-erbB-2, since the latter exhibited a stronger staining.

A parallel section to the conventional IHC stains presented in Fig. D.3.1.4-1 c, d was simultaneously incubated with Her 2 (Ho) and CK 7 (Tm) as described in section C.3, and a fraction providing a characteristic staining pattern was analyzed by LA-ICP-MS. The black rectangle in Fig. D.3.1.4-1 c highlights an area in the breast cancer tissue, which is Her 2 negative on the right side, and Her 2 positive on the left side. These microscopic findings are very well represented by the LA-ICP-MS image displayed in Fig. D.3.1.4-1 c, proving the high selectivity of labeled primary antibodies, and an optimum performance of the LA-ICP-MS. Moreover, the existence of a positive cell group indicated by arrows is identified in the LA-ICP-MS image, as well as in the conventionally stained thin section. Concerning CK 7, a structure in the

99 Part D: Results and Discussion tissue exhibiting highly pronounced expression of the antigen was encircled in black (Fig. D.3.1.4-1 d), and was identified by both detection methods. The results indicate that washout times of the ‘two volume cell’ were suited for imaging applications employing the New Wave 213 laser ablation system.

Thus, this experiment verifies the sustained selectivity of the antibodies during labeling, and that lanthanide labeled tumor markers employed in this work are fully applicable for differentiation of positive and negative tissues in IHC analysis with LA-ICP-MS detection. Furthermore, the lower level of expression of CK 7, which was indicated by the conventional IHC staining, is found in LA-ICP-MS analysis of the tissue as well, since Tm is detected at lower intensities than Ho.

100 BAM-Dissertationsreihe Part D: Results and Discussion

Figure D.3.1.4-1 Immunohistochemical staining of 3 µm breast cancer tissue sections, positive for a MUC 1 (scale bar: 2 mm), c Her 2 (left), d CK 7 (left). b Immunohistochemical staining of 3 µm palatine tonsil tissue section, negative for MUC 1, scale bar: 2 mm. A parallel thin section was simultaneously incubated with Her 2 (Ho) and CK 7 (Tm), and analyzed by LA-ICP-MS. The resulting images are displayed in c, and d, respectively. Laser parameters c, d: Laser spot size 200 µm, scan speed 150 µm s-1, repetition rate 10 Hz, laser energy 35 %. Characteristic microscopic findings are highlighted by black frames. Figure adapted from reference 185, courtesy of Analytical Chemistry.

As already mentioned before, a prerequisite for analyses is the high selectivity of antibodies, which results in a sufficient signal-to-background ratio to distinguish positive from negative controls. These two parameters were carefully evaluated. For the deterimation of signal-to-background ratios, the tissue was cut at 5 µm, and simultaneously incubated with labeled antibodies, as described in the experimental section. Examples of single line scans of breast cancer tissue sections (positive controls) are presented in Fig. D.3.1.3-1 for MUC 1, and in Fig. D.3.1.4-2 a, b for

101 Part D: Results and Discussion

Her 2 and CK 7, respectively. Single line scans of identically processed palatine tonsil tissue (negative controls, Fig. D.3.1.4-2 c–e) were analyzed during the same ICP-MS sequence as positive controls. Holmium and Tm intensity time profiles shown in Fig. D.3.1.4-2 a, b and c, d were from the same single line scans due to a parallel incubation of Her 2 (Ho) and CK 7 (Tm). The line scans shown in Fig. D.3.1.4-2 e and in Fig. D.3.1.3-1 were from parallel thin cuts of positive (breast cancer) and negative (palatine tonsil) tissue blocks.

In the case of Her 2 (Ho) negative controls displayed in Fig. D.3.1.4-2 c, measured intensities were hardly distinguishable from background noise in the tissue (< 500 cps), verifying the fully retained high selectivity of lanthanide labeled tumor markers. The signal-to-background ratio was calculated from the mean signal of positive and negative controls, and was approximately 12 for Her 2 (Ho). For MUC 1 (Tb), the negative control resulted in ~ 2000 cps, and signal-to-background was approximately 40. Hence, the labeled MUC 1 was successfully applied to tissue sections with retained high selectivity. As has been mentioned in section C.3, CK 7 needed further purification prior to Tm labeling since it was provided as cell culture supernatant. However, during purification not all cell culture proteins might have been removed. This can explain why a slightly higher signal-to-background of approximately 6 is detected in the palatine tonsil tissue section compared to Her 2 and MUC 1. Nevertheless, negative controls were clearly distinguishable from positive controls when applying CK 7 (Tm).

102 BAM-Dissertationsreihe Part D: Results and Discussion

a b

c d

Figure D.3.1.4-2 Single line scans of 5 µm breast cancer tissue section a-b, and 5 µm palatine tonsil tissue section c-e. Her 2 (Ho) (a, c) and CK 7 (Tm) (b, d) were detected at laser spot size 200 µm, scan rate 150 µm s -1, repetition rate 10 Hz, laser energy 35 %. MUC 1 (Tb) (e) was detected at laser spot 200 µm, scan rate 200 µm s -1, repetition rate 20 Hz, laser energy 35 %. The positive control corresponding to (e) is given in Fig. D.3.1.3-1 g. Figure adapted from reference 185, courtesy of Analytical Chemistry.

103 Part D: Results and Discussion

D.3.2 Multiplex IHC As has been mentioned before, the multi element capabilities of ICP-MS can be applied for the development of a multiplex assay. In this work, lanthanide labeled antibodies were employed for IHC with LA-ICP-MS as a new detection tool. By exploiting the multiplex capabilities of LA-ICP-MS, tumor markers can be directly detected and compared within the same tissue section, which facilitates standardization, and reduces analysis time. An independent assessment of several tumor markers on the same tissue section is not possible by routine IHC. Even the use of three different dyes does not solve this problem due to an overlap of dyes for co-localized tumor markers. Hence, time consuming subsequent staining procedures of parallel thin sections are commonly applied in IHC.

D.3.2.1 Multiplexed Detection of Her 2, CK 7, and MUC 1 in Breast Cancer Tissue For the multiplexed evaluation of tumor markers already discussed in section B, three lanthanide labeled primary antibodies – Her 2 (Ho), CK 7 (Tm), and MUC 1 (Tb) – were simultaneously applied for the first time on breast cancer tissue with LA-ICP-MS detection. For an optimized multiplex analysis a 200 µm laser spot size was used to guarantee a sufficient intensity for all three elements. Rastering of the whole tissue section was performed employing line by line scanning. Ablated line scans in the tissue were 180 µm wide. Thus, distribution of tumor markers was monitored at a spatial resolution of 180 µm, and 200 µm s-1 scan speed. The results are presented in Fig. D.3.2.1-1 a–c. All three tumor markers were detected above background level with signal-to-background ratios ≥ 6. Thus, they are evaluated as being positive in this breast cancer tissue section, as confirmed also by immunohistochemical staining in Fig. D.3.1.4-1. Details of the tissue geometry seen in the conventional IHC are very well represented by the LA-ICP-MS image, although resolution is about a factor of 100 inferior to light microscopy. For example, the tumor marker MUC 1 is negative on the upper left rim of Fig. D.3.2.1-1 c (highlighted by a black rectangle), where Tb signal is detected at background level. This observation is in good agreement with the stained thin section displayed in Fig. D.3.1.4-1 a, where MUC 1 staining was also negative (left rim, black rectangle).

104 BAM-Dissertationsreihe Part D: Results and Discussion

In the investigated tissue, Ho and Tm signals yielded up to 3000 cps, indicating an overexpression of tumor markers Her 2 and CK 7. Interestingly, MUC 1 showed a much higher intensity in this breast cancer tissue by a factor of ~ 40, compared to Her 2 and CK 7. This effect cannot be explained by a different ICP response since all analytes monitored were monoisotopic lanthanides, which should behave similar within the ICP. The higher sensitivity of MUC 1 (Tb) could be explained either by a more pronounced expression of mucin 1 within the tissue, or by a higher labeling degree per antibody. Therefore, the average labeling degree for MUC 1 (Tb) was determined by nESI-Q-TOF-MS (results are presented in the appendix), which is in accordance to other antibodies investigated so far (two labels per antibody).57 Thus, mucin 1 most likely exhibits a higher expression level as c-erbB-2 and cytokeratin 7 in the examined breast cancer tissue. Results obtained by LA-ICP-MS indicate a more pronounced expression for MUC 1, than diagnosed from the conventional IHC stains displayed in Fig. D.3.1.4-1. Hence, a deeper insight might be attained by LA-ICP-MS, which provides a larger dynamic range from a few hundred up to 1.5 × 105 cps in this case. Under optimized conditions for both sample preparation and measurement a direct comparison of up to three different tumor markers within a single 5 µm thin tissue section was accomplished using LA-ICP-MS as a detection tool. Due to subsequent staining procedures of parallel thin sections a comparable experiment is not feasible in conventional IHC.

In this novel experiment the simultaneous detection of three different breast cancer markers was accomplished in a single tissue section. Up to now, three different thin sections had to be subsequently processed to receive this information and usually, these thin sections are not even parallel cuts. The risk is high that those parallel cuts, which are processed at different days, and most likely by another technician, are not comparable to each other due to varying staining conditions. In this regard, the application of LA-ICP-MS is highly advantageous, since sample preparation is completed within the same day, and by the same individual. The direct comparability is of great benefit for standardization of the results in IHC. Furthermore, the sample throughput might be increased if even more parameters are monitored simultaneously, which is faster and more efficient than the performance of subsequent staining protocols. In cancer diagnosis, time may play a critical role and hence, time saving methods are urgently needed.

105 Part D: Results and Discussion

Figure D.3.2.1-1 LA-ICP-MS images of 5 µm breast cancer tissue, incubated with a Her 2 (Ho), b CK 7 (Tm), and c MUC 1 (Tb). Laser parameters: Laser spot size 200 µm, scan speed 200 µm s -1, repetition rate 20 Hz, laser energy 35 %. Total analysis time: 1 h. Figure adapted from reference 185, courtesy of Analytical Chemistry.

Since analysis of MUC 1 exhibited signal intensities of up to 1.5 × 105 cps at 200 µm laser spot size, imaging of the breast cancer tissue was feasible at even higher spatial resolution. A parallel tissue section was incubated simultaneously with Her 2 (Ho), CK 7 (Tm), and MUC 1 (Tb), and analyzed by LA-ICP-MS at a laser spot size of 50 µm. At this high spatial resolution, intensities for Ho and Tm were indistinguishable from background, but Tb was detected at up to 1.5 × 104 cps. An image of MUC 1 distribution is displayed in Fig. D.3.2.1-2. The LA-ICP-MS image was inserted into the corresponding microscopic photograph of the breast cancer tissue section. Again, the high selectivity of the labeled antibody is proven, since Tb intensities were detected at background level on the right side of the tissue, where mucin 1 is most likely expressed at very low levels.

106 BAM-Dissertationsreihe Part D: Results and Discussion

Figure D.3.2.1-2 LA-ICP-MS images of 5 µm breast cancer tissue, incubated with MUC 1 (Tb). Laser parameters: Laser spot size 50 µm, scan speed 25 µm s-1, repetition rate 10 Hz, laser energy 35 %.

D.3.2.2 Comparison of MUC 1 with Cu and Zn Distribution in Breast Cancer Tissue The distribution of metals in tissue sections has already been monitored by LA-ICP-MS, as discussed in section B.1. This information can be obtained in addition to the multiplexed detection of tumor markers in the tissue. Copper and zinc are essential trace elements and have therefore been selected here for detection in the breast cancer tissue. Moreover, recent clinical studies have pointed out the interdependency of lower copper levels with regulation of tumor growth.186 There are several anti-copper drugs under investigation, and zinc was tested amongst others in animal mouse models.186 Furthermore, Zoriy et al.187 found copper accumulation around a human brain tumor by LA-ICP-MS imaging.

The results of this experiment are shown in Fig. D.3.2.2-1 a–c, and were conducted on a parallel thin cut, which was identically processed during sample preparation as the tissue shown in Fig. D.3.2.1-1 a–c. However, since more isotopes were monitored within the same time frame in LA-ICP-MS measurements, sensitivity

107 Part D: Results and Discussion decreased by approximately 500 cps for Ho and Tm, which was significant for Her 2 and CK 7 monitoring since they were less abundant as the tumor marker MUC 1 (results are not shown here).

Copper and zinc are naturally abundant metals and were almost homogeneously distributed throughout the thin section except for a region on the left rim of the tissue, where an enrichment of copper, and also a slight increase in zinc was observed. For the direct comparison of MUC 1 and copper distribution, Fig. D.3.2.2-1 a was merged with the corresponding Tb image (not shown here, similar to Fig. D.3.2.1-1 c), resulting in Fig. D.3.2.2-1 b. Copper distribution is displayed in green, and MUC 1 positive cells are shown in red. In accordance to the immunohistochemical stain in Fig. D.3.1.4-1 a, terbium signal decreased on the left rim of this tissue, as the tumor marker MUC 1 is expressed at a very low level here. A possible explanation of copper enrichment might be the existence of a tumor invasion zone at the edges of the actual tumor (since MUC 1 is negative here), as observed also by Zoriy et al.187

Metal distributions have already been measured in tissue samples.28 In this experiment cancer biomarker expression levels can be additionally visualized to naturally abundant metal distributions to see where cancer cells might have affected metal homeostasis. This could become an important tool to understand the role of metals during cancer development. Furthermore, it is of interest if these metals are related to specific metalloproteins. In principle this question might be answered in the future using the presented approach to label specific antibodies for detection of metalloproteins. The technique developed here can not only be applied for cancer biomarker detection, but analysis of any antigen of biological or biochemical interest, which shows a sufficient level of expression.

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Figure D.3.2.2-1 LA-ICP-MS images of 5 µm breast cancer tissue, incubated with 65 65 Her 2 (Ho), CK 7 (Tm), and MUC 1 (Tb). a Cu, b Color merge of Cu (green) and Tb (red), 66 -1 c Zn. Laser parameters: Laser spot size 200 µm, scan speed 200 µm s , repetition rate 20 Hz, and laser energy 35 %.

D.3.3 Summary In this work, the sample preparation of tissues including the labeling conditions of biomarkers, and laser ablation parameters, were elucidated for LA-ICP-MS of thin tissue sections. For this purpose, different tissue thicknesses were evaluated regarding their suitability for laser ablation. Furthermore, variations in antibody concentration and incubation time were optimized. Under optimum conditions, tissue samples were cut at a thickness setting of 5 µm, incubated with a primary antibody concentration of ~ 1 µg mL-1 for 3 hours, and analyzed at 200 µm laser spot size, 200 µm s-1 scan speed, 20 Hz repetition rate, and 35 % laser energy to yield good signal intensities. Compared to conventional IHC, antibody concentration was lower and hence, incubation times were longer. Nevertheless, the results of LA-ICP-MS imaging are in good agreement with IHC stained thin sections. Furthermore, the selectivity of labeled primary antibodies was successfully investigated.

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In a novel multiplex experiment, the simultaneous detection of up to three different tumor markers in breast cancer tissue was accomplished. For the first time, tumor markers were not detected on parallel cuts by means of subsequent staining protocols, but were comparable to each other on a single thin tissue section using LA-ICP-MS as a detection tool. A similar experiment is not feasible in standard IHC due to an overlap of organic dyes for co-localizied tumor markers. However, the development of highly sensitive labels, based on polymer tags or nanoparticle tags, might improve spatial resolution in LA-ICP-MS, provided the selectivity of the antibodies is sustained. Additionally, copper and zinc distributions were monitored in the breast cancer tissue, visualizing where cancer cells might have affected metal homeostasis.

Currently, resolution is still a factor of 10 to 100 inferior to light microscopy. By applying highly selective antibodies, as well as highly amplifying labels resolution of LA-ICP-MS might be enhanced down to the cellular level, which is desirable to compete with conventional IHC. Therefore, highly amplifying labels based on polymer tags46 or nanoparticle tags could be employed for IHC. However, it is a challenge for these tags to preserve the high selectivity and low nonspecific binding of antibodies in the tissue (refer to section F.3).

In the future, the development of assays with highly amplifying labels such as nanoparticles or polymertags is highly desirable to further reduce spatial resolution in LA-ICP-MS-based tissue imaging. Furthermore, the simultaneous application of an increased number of tumor markers will be of great benefit (i) to reduce the time needed until a tumor-identifying antigen-profile can be determined, (ii) costs, and (iii) the material consumed for analysis.

D.4 Iodine as an Elemental Label for Imaging of Single Cells and Tissue Sections by LA-ICP-MS For tumor diagnostics it is important to interpret the composition of tissues or even further, of cells and cell compartments. In histology, tissue components are often visualized by HE staining. Hematoxylin imparts a blue color to cell nuclei, whereas eosin stains most other components pink. Additionally, IHC is employed in cancer

110 BAM-Dissertationsreihe Part D: Results and Discussion diagnostics to assess the expression level and the intracellular localization of a tumor marker in tissue sections. A drawback of this method is the lack of standardization, which in part is due to the inherent difficulty of quantification and reproducibility.89 LA-ICP-MS is a promising tool to study protein distribution either by the detection of heteroatoms, or by metal tags.21 Furthermore, the labeling of proteins with iodine has been reported, and is applicable to electrophoresis and electro-blotting, owing to the covalent linkage.59, 60 Radioactive iodine has been first used by Hunter61 for labeling of hormones. Up to now, many other complex iodination agents have been applied,62- 65 but the use of a saturated iodine solution in potassium iodide is less laborious and inexpensive. Direct labeling of proteins with iodine occurs at the two ortho-positions of tyrosine and at histidine residues. These electrophilic aromatic substitutions require reactive iodine species like triiodide, which is formed in aqueous solution from - - iodide and elemental iodine. To a lower extent also polyiodide ions such as I5 and I7 are formed.

In a recent study of Wu et al.,82 laser microdissection ICP-MS was used for imaging of heteroelements in brain tissue at laser spot sizes from 30–4 µm. Nevertheless, spatial resolution has been inferior to light microscopy due to a loss of sensitivity when using small laser spot sizes. Therefore, important information on tissue morphology is lost, as compared to HE staining in conventional histology.

Information on protein distribution in cancer diagnostics is fundamental, but complementing quantitative data is urgently needed since alterations in the copy number of genes may play a role in the pathogenesis.86 However, an internal standard is a prerequisite to obtain quantitative data in ICP-MS in order to correct for fluctuations within the ICP, and for thickness inhomogeneities in tissue sections. Thus, an internal standard is needed, which should have a similar response to the ICP as the analytes monitored, i.e. mass differences should not be too large. Usually 13C is used as internal standard for laser ablation of thin sections,67, 73, 79 albeit lower or higher water content in the tissue leads to underestimation or overestimation of tissue thickness and therefore affects quantification approaches.67 Moreover, Todolí et al.188 reported on the different washout of formed gaseous species of carbon to that of sample aerosol. However, 13C internal standardization is not applicable to LA-ICP-MS-based IHC in this case, due to the organic coating of glass slides, which is needed during IHC sample preparation.

111 Part D: Results and Discussion

Iodine labeling was applied for single fibroblast cells and for thin tissue sections in LA-ICP-MS-based imaging and IHC for the first time. First results on the ability to provide information on tissue and single cell morphology, and the use of iodine as an internal standard for thin slices will be presented in the following sections.

D.4.1 Iodination of Fibroblasts In previous work it was demonstrated that whole proteomes can be efficiently iodinated,60 but it had not been applied to single cells or tissue sections. The iodination of fibroblast cells and of thin tissue sections was performed prior to analysis. Therefore, fibroblast cells were fixed on a microscopic glass plate and iodinated by treatment with KI3 solution and subsequent reduction by Na2S2O4. During iodination of fibroblast cells, a deep brown staining of cell nuclei was observed which started to spread out to the cytoplasm after 60 s. The brown color disappeared as soon as the reaction was stopped. Figure D.4.1-1 shows a photograph of the fibroblast cells prior to laser ablation, and the corresponding iodine distribution determined by LA-ICP-MS. Laser ablation was performed line by line with a 4 µm laser spot size, and 5 µm s-1 scan speed. The resulting single line scans were 6 µm wide, which demonstrates that with the applied irradiance, even the tails of the Gaussian laser pulse are still sufficient to ablate material. This effect is only observed for very thin samples as they are discussed here. Consequently, a distance of 6 µm was set between adjacent line scans. The surface plot of the iodine signal in Fig. D.4.1-1 allows to identify the cells on the glass slide and to differentiate between cytoplasm and nucleus, albeit tailing of the iodine signal was observed due to ablated aerosol dispersion. Comparison of the photograph with the LA-ICP-MS image shows the highest iodine signal of up to 5 × 104 cps inside the cell nuclei, and lower intensities in the surrounding cytoplasm. Iodine was detected with high sensitivity at a laser spot size of only 4 µm, which is approximately one-tenth of the size of the imaged fibroblast cell nucleus (see Fig. D.4.1-1). Hence, the method described herein is highly sensitive and the resolution achieved is capable to analyze single cells and even cell nuclei by LA for the first time. The resolution achieved in these first experiments permits the detection of even smaller cells, and these experiments can open the door to apply LA as a new microscopic tool.

112 BAM-Dissertationsreihe Part D: Results and Discussion

Figure D.4.1-1 Lower part: Microscope photograph of fixed, iodinated fibroblast cells. Upper part: LA-ICP-MS image of iodine distribution. Laser parameters: laser spot size 4 µm, scan speed 5 µm s-1, repetition rate 5 Hz, laser energy 35 %. Figure adapted from reference 189, courtesy of Journal of Analytical Atomic Spectrometry.

D.4.2 Optimization of Tissue Labeling by Iodination The iodination of thin tissue sections has not been employed before and thus had to be optimized in this work. For this purpose, a palatine tonsil tissue, sectioned at a thickness setting of 5 µm was iodinated for 30 s, 60 s, and 90 s, respectively. The iodine intensity of the corresponding single line scans across the tissue are displayed in Fig. D.4.2-1. The iodine signal increased by a factor of ~ 3 by extending the incubation time from 30 s to 60 s. The signal intensity was less enhanced (factor of ~ 2 only) by increase of incubation time to 90 s. Keeping in mind that iodine diffuses out to the cytoplasm after 60 s, which was observed during iodination of fibroblast cells, an iodine incubation time of 60 s was selected for all further experiments.

113 Part D: Results and Discussion

4x107

90 s 7 3x10 60 s 30 s

2x107 127I [cps] 127I

1x107

0 0 5 10 15 20 25 30 35 40 time [s]

Figure D.4.2-1 Iodination of a palatine tonsil tissue section for 30 s (red line), 60 s (blue line), and 90 s (black line), respectively. Laser parameters: laser spot size 200 µm, scan speed 200 µm s-1, repetition rate 20 Hz, laser energy 35 %. Figure adapted from reference 189, courtesy of Journal of Analytical Atomic Spectrometry.

D.4.3 Iodination of Liver Biopsy Tissue A photograph of a fraction (about 500 × 800 µm in size) of a 3 µm thick HE stained liver biopsy tissue is depicted in Fig. D.4.3-1 a. The photograph was taken with the camera installed inside the NWR 213 laser ablation system, and therefore the quality is lower than typical microscopic pictures. A second parallel 5 µm thin section was treated with iodine as described above, and analyzed by LA-ICP-MS. The tissue was ablated line by line at 4 µm laser spot size, and 5 µm s-1 scan speed. Single line scans were 6 µm wide and therefore, the distance between adjacent scans was set to 6 µm. As depicted in Fig. D.4.1-1, there is less iodine signal in the cytoplasm, which is represented by iodine background in Fig. D.4.3-1 b, c. On the contrary, the iodine peak in the single line scan presented in Fig. D.4.3-1 c is related to a cell nucleus, which obviously has a higher iodination level than the surrounding cytoplasm. The LA-ICP-MS image reveals a distribution of iodine in small but distinct areas, as illustrated in Fig. D.4.3-1 b. However, not all cell nuclei can be identified by comparison of the LA-ICP-MS image with the microscopic picture of the parallel

114 BAM-Dissertationsreihe Part D: Results and Discussion tissue section. Some iodine signals in Fig. D.4.3-1 b could be related to smaller cell nuclei which are absent in the HE stained section displayed in Fig. D.4.3-1 a. Nevertheless, one characteristic cell nucleus highlighted by a red square in Fig. D.4.3-1 a, and is represented by iodine signal in the same area of the tissue in Fig. D.4.3-1 b. This cell nucleus has a tetraploid set of chromosomes and is therefore enlarged, which has been observed in the liver before.190 Cell nuclei > 8 µm are visible in parallel sections, provided they are cut centered. Small cell nuclei < 8 µm are not visible in both the HE stained and the ablated tissue section since their size is smaller than the distance between parallel sections. Hence, only the use of ultra thin tissue sections permits a resolution down to the cell nucleus level.

By iodination of single cells or tissues prior to laser ablation, morphological information can be gained by LA-ICP-MS images, comparable to the information received by HE staining in light microscopy. The identification of a liver cell nucleus with a tetraploid set of chromosomes, and its discrimination to the surrounding tissue was accomplished by iodination and LA-ICP-MS imaging. Thus, iodine can in principle be used to measure cells in tissue samples, and therefore was applied as an internal standard to correct for tissue thickness inhomogeneities in LA-ICP-MS (refer to chapter D.4.4).

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Figure D.4.3-1 a HE stain of liver biopsy. b Iodine signal, single line scan of a 5 µm thin iodinated liver biopsy. Laser parameters: laser spot size 4 µm, scan speed 5 µm s-1, repetition rate 5 Hz, laser energy 35 %. c LA-ICP-MS image of iodine distribution in a 5 µm thin section of an iodinated liver biopsy. The red square in (a) highlights a tetraploid liver cell nucleus, and the corresponding iodine signal is shown in (c). The red line in (b) illustrates the single line scan and scan direction in (c). Figure adapted from reference 189, courtesy of Journal of Analytical Atomic Spectrometry.

D.4.4 A New Internal Standard for Tissue Sections Iodination was applied to 2, 5, and 8 µm thick sections of palatine tonsil tissue in order to examine the dependence of iodine signal intensity on tissue thickness and hence, on the quantity of cells. Single line scans of the tonsil tissue sections were performed at 100 µm laser spot size, and 100 µm s-1 scan speed. Results of the intensity measurement of 127I are depicted in Fig. D.4.4-1 for three single line scans of parallel thin cuts of the palatine tonsil tissue. According to these results, iodine

116 BAM-Dissertationsreihe Part D: Results and Discussion signal increased with increasing tissue thickness from ~ 2 × 107 cps to ~ 4 × 107 cps to ~ 6 × 107 cps for each 3 µm in tissue thickness and thus is directly proportional to the sample thickness. Hence, iodine proves to be well suited as internal standard to correct for tissue inhomogeneities in laser ablation of thin cuts.

1.0x108

8 µm 5 µm 8.0x107 2 µm

6.0x107

7 127I [cps] 127I 4.0x10

2.0x107

0.0 0 5 10 15 20 25 30 time [s]

Figure D.4.4-1 Single line scans of iodinated palatine tonsil thin slice, sectioned at 2 µm (red line), 5 µm (blue line) and at 8 µm (black line) thickness. Laser parameters: laser spot size 100 µm, scan speed 100 µm s-1, repetition rate 20 Hz, laser energy 35 %. Figure adapted from reference 189, courtesy of Journal of Analytical Atomic Spectrometry.

As already mentioned before, 13C is usually applied as internal standard for laser ablation of thin sections,67, 73, 79 but this method affects quantification approaches.67 Alternatively to 13C, iodine was tested as an internal standard for LA-ICP-MS on tissue sections, according to the observation that iodine signal is proportional to tissue thickness. For this purpose, a breast cancer tissue section was incubated with Her 2 (Ho) and CK 7 (Tm) prior to iodination. The iodination of proteins is covalent, which in principle can have an effect on antibody binding. Thus, a breast cancer tissue incubated with MUC 1 (Tb) was analyzed by a single line scan prior to and after iodination. However, signal intensities of Tb did not decrease after iodination (results are not shown here). Thus, it can be concluded that the applied method did not affect antibody binding and can be employed further on.

117 Part D: Results and Discussion

The tissue, which had been first incubated with Her 2 (Ho) and CK 7 (Tm), followed by iodination, was ablated line by line at 200 µm laser spot size, and 150 µm s-1 scan speed. Since the tissue sections were 5 µm thin, they were completely ablated during the measurements together with the polymer substrate material. Resulting LA-ICP-MS images, visualizing holmium, thulium, and iodine distribution in the breast cancer tissue section, are presented in Fig. D.4.4-2 a–c. Holmium and Tm signals were corrected for variations in tissue thickness by normalization to 127I. Tumor marker distribution, normalized to 127I, is illustrated in Fig. D.4.4-2 d–e. In the center of the thin section displayed in Fig. D.4.4-2 a, indicated by a black arrow, the expression of Her 2 seems to be reduced. Instead, this effect can be explained by tissue inhomogeneities and can be corrected for by normalization to the measured iodine signal, which shows a lower intensity in this region, as illustrated in Fig. D.4.4-2 d. Furthermore, the Ho signal in the breast cancer tissue is higher than the corresponding Tm signal (see Fig. D.4.4-2 d–e), indicating a higher expression level of Her 2 in comparison to CK 7. These findings are in good agreement with routine IHC stains of parallel thin sections (refer to section D.3).

Tissue sections for LA-ICP-MS measurements were mounted onto Superfrost Plus slides. The surface coating minimizes lanthanide background caused by glass ablation at optimized laser energy. For two line scans of the LA-ICP-MS image shown in Fig. D.4.4-2 a–b, signals for Ho and Tm were detected outside the tissue. This was probably due to a damaged surface coating in this area, so that glass was ablated as well. For glass slides with intact surface coating and optimized laser energy, glass ablation is held at a minimum. This can be controlled by monitoring other lanthanide elements, e. g. europium. A LA-ICP-MS image of 153Eu distribution is given in Fig. D.4.4-2 f. By normalizing Ho and Tm to corresponding 153Eu signals, data can be corrected for glass ablation, as shown in Fig. D.4.4-2 g–h. Hence, Eu is a good indicator for glass ablation and can be used for correction of this effect, if necessary. Additionally, Eu can be monitored during optimization to avoid glass ablation, which can cause elevated background for many elements of interest.

118 BAM-Dissertationsreihe Part D: Results and Discussion

Figure D.4.4-2 a Her 2 (Ho) distribution in 5 µm thin breast cancer tissue section, b CK 7 (Tm) distribution in 5 µm thin breast cancer tissue section. Laser parameters: laser spot -1 size 200 µm, scan speed 150 µm s , repetition rate 10 Hz, laser energy 35 %. c Corresponding iodine distribution. d Iodine normalization for Her 2 to correct for tissue thickness inhomogeneities. e Iodine normalization for CK 7 to correct for tissue thickness 153 inhomogeneities. f Eu signal. g Europium normalization for Ho to correct for glass ablation. h Europium normalization for Tm to correct for glass ablation. Figure adapted from reference 189, courtesy of Journal of Analytical Atomic Spectrometry.

D.4.5 Summary Iodination of fibroblast cells and of thin tissue sections had not been applied before and was optimized in this work. The method resulted in elemental staining of the cell nuclei, which was represented by distinct iodine signals in LA-ICP-MS measurements. A laser spot size of 4 µm was employed for ablation of fibroblasts and liver biopsy tissue. With this new method the resolution of single cell nuclei in

119 Part D: Results and Discussion fibroblast cells and in a liver biopsy tissue section was achieved, wherein a physiologic example for tetraploidy in liver cells was identified. Thus, iodine could in principle be used to measure single cells in tissue samples. Significantly smaller spot sizes of approximately 50 nm are claimed for tip-enhanced laser desorption/ablation,191 but so far only opaque substrates could be analyzed by this method.

Furthermore, a palatine tonsil tissue was sectioned at 2, 5 and 8 µm, and iodine signal increased with increasing tissue thickness. Consequently, iodine could be employed as internal standard to correct for tissue thickness inhomogeneities in a breast cancer tissue section, where Her 2 and CK 7 distribution was investigated. Furthermore, Eu normalization was used to correct for lanthanide background due to glass ablation in rare cases of damaged surface coating. Future work could involve the production of quantitative data for tumor marker distribution in tissue sections, applying iodine as an internal standard.

D.5 LA-ICP-MS Detection of Platinum-bound Proteins separated by 1D- and 2D-Gel Electrophoresis The metabolite profiling and quantification of metal containing drugs has been accomplished by ICP-MS, as has been reviewed by Gammelgaard and Jensen.192 Cisplatin is a prominent example of a metal containing drug, which has been successfully used for the treatment of solid tumors96 due to its ability to bind and distort the DNA in the nucleus.98 Moreover, proteins are likely to interact with cisplatin as well.99 The separation of proteins by gel electrophoresis with subsequent tryptic digestion and detection by organic MS is a powerful tool for the identification of proteins, but has not yet been applied to a kidney cell culture due to the very high number of proteins usually being separated. However, the combination of a metal detector such as LA-ICP-MS is helpful to identify only those proteins, which are conjugated with Pt.

For LA-ICP-MS, the experimental limit of detection of a standard Pt solution, which was pipetted and dried on a nitrocellulose membrane, was 2.5 pg Pt (0.5 µL of a -1 5 ng g Pt standard solution in 1 % HNO3). Nevertheless, electroblotting and the subsequent laser ablation of platinated proteins on a membrane are not applicable in

120 BAM-Dissertationsreihe Part D: Results and Discussion this case. This is due to contamination caused by the Pt electrodes employed during blotting following standard protocols with the Trans-blot SD semi-dry transfer cell by BioRad (Munich, Germany), which have a larger surface as the electrodes employed for gel electrophoresis and thus, increase Pt background by a factor of ten. Consequently, electroblotting hampers the analysis of low abundant proteins, e.g. in a cell lysate due to increased LODs. The application of carbon electrodes might solve this shortcoming.

As an alternative, proteins can be directly detected in the gel upon separation by SDS-PAGE.51 Since SDS is a protein denaturing agent, special attention has to be paid on the possibility of metal losses during gel electrophoresis, and staining of metal-protein complexes.193 Furthermore, splintering of the gel during laser ablation must be avoided. In the following chapter, the optimization of SDS-PAGE and LA-ICP-MS-based detection of metalloproteins is presented.

Different methods were tested for gel drying, such as air drying, the drying of the gel between filter papers, and the incubation of the gel in glycerol for 2 minutes with subsequent heating in an oven at 75 °C for 2 h. The latter procedure produced gels, which were stable during LA-ICP-MS measurements, in combination with a clean ablation. Hence, this drying method was used for all gels employed in this work.

D.5.1 Optimization of 1D-SDS-PAGE for LA-ICP-MS Secondary and non-disulfide-linked tertiary structures are denatured by SDS, whereas disulfide-bonds are usually cleaved by heating the sample to 90 °C in the presence of reducing agents like beta-mercaptoethanol (BME). However, BME can also disrupt the coordination of platinum to proteins. Furthermore, the presence of a free thiol group likely imparts a changed reactivity on the investigated proteins prior to GE and thus, proteins which had not been platinated before might react with free platinum. Therefore, five standard proteins (human serum transferrin (TF), human serum albumin (HSA), carbonic anhydrase from bovine erythrocytes (CA), horse heart myoglobin (MYO), and equine cytochrome c (CYT C)) were incubated with cisplatin and separated by 1D-SDS-PAGE as described in section C.5. To investigate the influence of BME, one replicate each was treated with and without the reagent, respectively. After drying the gel with glycerol, it was analyzed by LA-ICP-MS

121 Part D: Results and Discussion

(Element XR coupled to LSX-213, laser ablation cell: ETH Zürich) at 200 µm laser spot size, and 30 µm s-1 scan rate. Corresponding single line scans, together with the CBB stain, are displayed in Fig. D.5.1-1. The proteins were detected with a signal-to- background ratio of 100 for 195Pt when BME was absent, and a signal-to-background ratio of 60 was achieved in the presence of BME. For the latter gel, an additional high 195Pt signal was detected at the running front, which might stem from small negatively charged Pt-complexes, which were disrupted from proteins. In contrast, a significantly lower signal was detected for 195Pt at the running front in the absence of BME. Taking into consideration the overall higher Pt intensities in this sample, the Pt-protein complexes are likely more stable when BME is omitted and hence, these conditions were applied further on.

Since it was reported in the literature that staining of the gels might induce metal losses,193 special attention was paid to this concern. The five standard proteins, incubated with 60 µM cisplatin, were individually run in duplicate in a 1D-SDS-PAGE. One half of the gel was stained with CBB, the other half was left unstained prior to LA-ICP-MS (200 µm laser spot size, 30 µm s-1 scan rate). The results are presented in Fig. D.5.1-2. For the high molecular weight proteins like TF and HSA (79 kDa and 67 kDa, respectively), and even for CA (29 kDa), no metal losses were detected since the 195Pt intensities of stained and unstained gels were very similar. However, 195Pt intensities of the low molecular weight proteins MYO and CYT C (17 kDa and 12 kDa, respectively) were reduced in case of the CBB stained gel, and it is most likely that the staining induced Pt loss. Nevertheless, the staining of gels is essential for the analysis of whole gels, especially for large 2D gels, since their complete ablation is highly time consuming. By the application of a gel stain, certain areas of interest can be selected for analysis, which can be completed in a significantly shorter period of time. Although Pt signal was reduced for lower molecular weight proteins, their absolute intensities were still sufficient, and a good signal-to-background ratio (S/N: 100–250) was achieved. Thus, 2D gels for the separation of platinated proteins were CBB stained for LA-ICP-MS to reduce analysis time.

122 BAM-Dissertationsreihe Part D: Results and Discussion

Mixture of 5 proteins: TF, HSA, CA, MYO, CYT C

CA-Pt MYO-Pt TF-Pt CYT-Pt HSA-Pt + BME - BME500 ng 500 ng 500 ng 500 ng 500 ng

TF-Pt HSA-Pt

CA-Pt

MYO-Pt CYT-Pt

Scan length

Figure D.5.1-1 1D-SDS-PAGE of five standard proteins (human serum transferrin (TF), human serum albumin (HSA), carbonic anhydrase from bovine erythrocytes (CA), horse heart myoglobin (MYO), and equine cytochrome c (CYT C)) incubated with 60 µM cisplatin, 500 ng protein per well. LA-ICP-MS in single line mode (200 µm laser spot size, 30 µm s-1 scan speed, 20 Hz repetition rate, 100 % energy). Heating of the sample with BME (blue line scan) versus the omission of BME (green line scan). The peak at the end of the running front is indicated in red.

123 Part D: Results and Discussion

Unstained gels

500 ng TF-Pt 500 ng HSA-Pt 500 ng CA-Pt 500 ng MYO-Pt 500 ng CYT-Pt 195Pt, cps 79 kDa 195Pt, cps 67 kDa 195Pt, cps 29 kDa 195Pt, cps 17 kDa 195Pt, cps 12 kDa 200000 80000 120000 100000 50000 100000 80000 40000 150000 60000 80000 60000 30000 40000 60000 100000 40000 20000 40000 20000 50000 20000 20000 10000 0 0 0 0 0 0123 4 01234 01 234 01234 01234 scan length [cm] scan length [cm] scan length [cm] scan length [cm] scan length [cm]

Coomassie Brilliant Blue-stained gels

500 ng TF-Pt 500 ng HSA-Pt 500 ng CA-Pt 500 ng MYO-Pt 500 ng CYT-Pt

195Pt, cps 195Pt, cps 195Pt, cps 195Pt, cps 195Pt, cps 80000 120000 100000 50000 200000 100000 80000 40000 150000 60000 80000 60000 30000 40000 60000 100000 40000 20000 40000 50000 20000 20000 20000 10000 0 0 0 0 0 01234 01234 01234 01234 01234 scan length [cm] scan length [cm] scan length [cm] scan length [cm] scan length [cm]

Figure D.5.1-2 Comparison of CBB stained versus unstained conditions of five standard proteins incubated with cisplatin. 500 ng of each protein per well. The lower molecular weight proteins are indicated. Laser parameters: 200 µm laser spot size, 30 µm s-1 scan speed, 20 Hz repetition rate, 100 % energy.

D.5.2 Identification of Platinated Protein Spots in 2D-GE by LA-ICP-MS To verify that the optimized conditions for 1D-SDS-PAGE were applicable to the second dimension, the five standard proteins employed before were separated in a 2D-GE (IEF + SDS-PAGE). The gels were silver stained, and one replicate was analyzed by LA-ICP-MS. For these measurements, the gel was cut into three pieces containing (i) TF + HSA, (ii) CA, and (iii) MYO + CYT C. Each piece was separately ablated by adjacent single line scans at 200 µm laser spot size, and 100 µm s-1 scan rate. The spots representing the five standard proteins are highlighted by red rectangles in the silver stained gel in Fig. D.5.2-1. The LA-ICP-MS images of the three pieces are in good agreement with the results obtained from the silver stained gel, as is demonstrated in Fig. D.5.2-1.

124 BAM-Dissertationsreihe Part D: Results and Discussion

Figure D.5.2-1 2D-GE (IEF + SDS-PAGE) of five standard proteins (each 500 ng). Comparison of silver stained gel with a parallel silver stained gel, which was subsequently analyzed by LA-ICP-MS. LA-ICP-MS images (adjacent single line scans at 200 µm laser spot size, 100 µm s-1 scan speed, 20 Hz repetition rate, and 100 % energy) are correlated with the silver stained gel. MW = Molecular weight. pI = isoelectric point.

The compatibility of the optimized method with 2D (IEF + SDS-PAGE)-GE had been investigated and thus, a cell lysate of platinated proteins was analyzed by LA-ICP-MS. Renal proximal tubule epithelial cells (RPTECs) obtained from pig kidney154 were incubated with 1mM cisplatin prior to cell lysis. A total amount of 50 µg of the cell culture cytosolic protein extract was applied for 2D-GE separation. Again, two replicates were produced: one silver stained gel for comparison, and one CBB stained gel for LA-ICP-MS measurements (single line scans every 500 µm; 200 µm laser spot size, 100 µm s-1 scan speed, 20 Hz repetition rate, and 100 % energy). Both gels are depicted in Fig. D.5.2-2. The 2D gel was cut into five sections, and each piece was separately analyzed. As a result, Pt signals in the LA image are in good agreement with protein spots in the stained gels. However, the intensity of

125 Part D: Results and Discussion the dye does not correlate with the Pt signal intensity in all cases. Hence, even a protein of low abundance might be heavily platinated and might play a key role in Pt metabolism. Additionally, the Pt spots are not as sharp as their stained counter parts. This is most likely due to longer washout times of the laser ablation chamber, which has a rather large volume for imaging applications, quite contrarily to the ‘two volume cell’. Nevertheless, it was the only available cell applicable to larger samples at the time of the analysis.

Figure D.5.2-2 2D (IEF + SDS-PAGE)-GE of renal proximal tubule epithelial cells (RPTECs) obtained from pig kidney. Total amount of the cell culture cytosolic proteins: 50 µg. Silver stained gel in comparison to CBB stained gel. The latter was analyzed by LA-ICP-MS (single line scans every 500 µm; 200 µm laser spot size, 100 µm s-1 scan speed, 20 Hz repetition rate, and 100 % energy). MW = Molecular weight. pI = isoelectric point.

D.5.3 Summary The analysis of 1D and 2D gels of standard proteins and protein extracts from cell lysates, both after treatment with cisplatin, was conducted by LA-ICP-MS after

126 BAM-Dissertationsreihe Part D: Results and Discussion optimum drying conditions were applied. With the LA-ICP-MS method employed in this work, the analysis of 2D gels was accomplished, bearing in mind the assumed loss of Pt for low molecular weight proteins owing to the staining protocol. However, Pt loss was minimized by omitting the reagent BME during gel electrophoresis. The method developed herein enables the identification of platinated protein spots in a straightforward way. In future work, these protein spots will be excised and digested in a parallel experiment for nLC-ESI-MS/MS analysis in order to identify proteins which interacted with cisplatin.

D.6 Imaging of Metal Distribution in Rat Kidneys by LA-ICP-MS The antitumor agent cisplatin is employed in more than half of the oncologic treatments.95 However, about one third of the patients suffer from nephrotoxic side- effects of the drug.101 The kidney is the main route for cisplatin excretion, where it accumulates to a greater extent than in other organs. Thus, toxicity is related to Pt accumulation, and information on the metal distribution in kidneys is beneficial to understand the cisplatin-induced nephrotoxicity. This may help to improve oncologic treatments and to reduce side-effects. A methodology for LA-ICP-MS-based bioimaging of 3 µm thin FFPE kidney tissues obtained from rats treated with pharmacologic doses of cisplatin has been developed and will be presented in this chapter. The accumulation of Pt, and the distribution of Cu and Zn were monitored after renal damage had taken place. The resolution achieved (8 µm) is sufficient to study metal accumulation in renal substructures. Furthermore, the protective effect of cilastatin on cisplatin nephrotoxicity was investigated. Recently, it was demonstrated that cilastatin reduces cisplatin nephrotoxicity in renal epithelial cell models.194

D.6.1 Imaging of Platinum Distribution

D.6.1.1 Optimization In this work, the 195Pt isotope is presented for Pt imaging. 194Pt was additionally monitored, showing the same elemental distribution.

Platinum in the tissue is mainly covalently linked to DNA98 or proteins195 and thus, should be unaltered by the applied sample preparation methods. The effect of dewaxing FFPE tissue sections was investigated on a kidney slice from a rat treated

127 Part D: Results and Discussion with 16 mg kg-1 bodyweight (bw) cisplatin, compared to a half of a parallel, paraffin- embedded thin section. Both thin sections were analyzed with 100 µm laser spot size, 150 µm s-1 scan speed, 20 Hz repetition rate, and 33 % laser energy. The analysis of a whole tissue section was accomplished in three hours. The resolution attained allows an excellent visualization of the morphological features cortex and medulla, as demonstrated in Fig. D.6.1.1-1 a, b. Images of dewaxed and of paraffin- embedded tissues exhibit similar metal distribution and 195Pt signal intensities. Resolution in the latter image seems to be slightly lower, which is probably due to paraffin melting during the ablation process. Thus, dewaxed tissue sections were used further on to achieve the highest attainable spatial resolution.

Reproducibility was studied by analyzing another parallel dewaxed slice of the same kidney at 100 µm laser spot size, 150 µm s-1 scan speed, 20 Hz repetition rate, and 33 % laser energy (see Fig. D.6.1.1-1 c). The Pt distribution pattern and signal intensities were similar to the thin section presented in Fig. D.6.1.1-1 a.

The resolution attained for a whole tissue slice is a compromise of analysis time and laser ablation parameters, which need optimization. For this purpose, a kidney section from a rat treated with a pharmacologically relevant dose of 5 mg kg-1 bw cisplatin was analyzed at 80 µm laser spot size, and 100 µm s-1 scan speed, in comparison to a parallel slice analyzed at 100 µm laser spot size, and 150 µm s-1 scan speed. The corresponding LA images are illustrated in Fig. D.6.1.1-1 d, e. Analysis time at 80 µm laser spot size was five hours. During that time instrumental drift of the ICP-MS and the laser might occur. However, since the laser parameters were optimized to complete ablation of the tissue, a drift of laser fluence has only a minor effect on the total tissue mass ablated. Instrumental drift of the ElementXR accounts for 3 % RSD as stated by the manufacturer, and is therefore negligible considering the sum of squares to calculate the entire uncertainty budget, which is mainly affected by sample inhomogeneities. On the other hand, an excellent resolution for Pt imaging of a whole rat kidney tissue was attained in a shorter analysis time (3 h) with100 µm laser spot size. Consequently, these settings were applied for all further analyses of whole tissue sections, since the resolution achieved allows the visualization of Pt distribution in the main renal structures.

128 BAM-Dissertationsreihe Part D: Results and Discussion

a b e

c e

Figure D.6.1.1-1 Optimization of LA-ICP-MS imaging of rat kidney thin tissue sections. The 195 isotope Pt is displayed in all images. a dewaxed sagittal kidney section from a rat treated -1 with 16 mg kg cisplatin; b paraffin-embedded parallel section to (a); c replicate of (a). LA parameters a–c, and e–f: 100 µm laser spot size, 150 µm s-1 scan speed, 20 Hz, 33 % laser -1 energy; d dewaxed sagittal kidney section from a rat treated with 5 mg kg cisplatin, LA -1 parameters: 80 µm laser spot size, 100 µm s scan speed, 20 Hz, 33 % laser energy; e parallel section to (d); f dewaxed sagittal kidney section from a control rat. Figures adapted from reference 196, courtesy of Analytical Chemistry.

D.6.1.2 Evaluation In a control kidney section no significant Pt signal was detected (background < 500 cps), as illustrated in Fig. D.6.1.1-1 f. Figures D.6.1.1-1 b, e show the Pt distribution obtained by LA-ICP-MS in kidney sections from rats treated with 5 mg kg- 1 bw and 16 mg kg-1 bw cisplatin, respectively. Compared to the 5 mg kg-1 sample, 195Pt intensities in the 16 mg kg-1 bw cisplatin treated sample were about one order of magnitude higher, reflecting the higher drug dose.

129 Part D: Results and Discussion

In the case of 5 mg kg-1 bw cisplatin, Pt accumulates mainly in the inner cortex and the corticomedullary region, but to a much lower extent in the medulla. These findings are quite contrary to the observations made for the 16 mg kg-1 bw cisplatin, where Pt accumulation extends from the cortex to the pyramidal structures of the medulla. This indicates an intoxication of the rat at higher cisplatin dosage. Remarkably, Pt intensities are about one order of magnitude lower in the medulla as in the cortex. This trend is in good agreement with the fact that the medulla is not significantly affected by renal damage.102 Instead, the Pt transporters are mainly located in the cortex,197 which explains the higher Pt accumulation detected therein.

In the 5 mg kg-1 bw cisplatin kidney section, Pt intensities of up to 106 cps for 195Pt were detected and thus, resolution could be significantly increased by reducing the laser spot size to 8 µm, and the scan speed to 10 µm s-1. Figure D.6.1.2-1 displays the Pt image of a small cortical area (~700 × 700 µm) selected on a thin section of a rat treated with 5 mg kg-1 bw cisplatin. A very high resolution was attained, discerning accumulation in the tubule cells, and lack of Pt in the glomerulus and in the tubule lumen (see red circles in Fig. D.6.1.2-1). These findings are in good agreement to a microscopic picture taken prior to laser ablation (glomerulus and lumen are indicated in red in Fig. D.6.1.2-1).

In the parallel HE stained sections, morphologic abnormalities indicated the toxic side-effects of cisplatin, which are illustrated in Fig. D.6.1.2-2 b, c. The healthy control (see Fig. D.6.1.2-2 a) was altered after administration of 5 mg kg-1 cisplatin, resulting in tubule swelling, loss of brush border membrane, epithelial vacuolization, hyaline casts (indicating protein aggregates), and cell debris detachment.

130 BAM-Dissertationsreihe Part D: Results and Discussion

-1 Figure D.6.1.2-1 Dewaxed sagittal kidney section from a rat treated with 5 mg kg cisplatin, LA parameters: 8 µm laser spot size, 10 µm s-1 scan speed, 20 Hz, 33 % laser energy. LA-ICP-MS image of 195Pt distribution (left) and microscopic picture taken prior to laser ablation (right). Glomerulus and lumen are indicated in red. Figures adapted from reference 196, courtesy of Analytical Chemistry.

131 Part D: Results and Discussion

a b

10x 10x

c d

60x 10x

Figure D.6.1.2-2 Histopathological study of HE-stained rat kidney sections for the evaluation of cisplatin-induced renal damage. a control, cortex in 10× magnification; b cisplatin 5 mg kg-1, cortex in 10× magnification; →: Protein casts formation, visualized as pink-stained, glassy cylinders inside the tubules lumen. Tubule swelling is marked with a blue circle. Cell nuclei are stained blue by hematoxylin, while most other cell components are pink-stained by -1 eosin. c cisplatin 5 mg kg , cortex in 60× magnification; *: vacuolization, →: cell debris detachment. d microscope image of a HE stained kidney section (cortex) from a rat treated with cisplatin 5 mg kg-1 + 150 mg kg-1 per day cilastatin in 10× magnification. Figures adapted from reference 196, courtesy of Analytical Chemistry.

D.6.1.3 Estimation of Total Pt Amount in Kidney Tissue Quantification in LA-ICP-MS is difficult for tissue samples due to the lack of appropriate matrix-matched standards. Furthermore, the established quantification strategies are commonly based on the use of 13C as an internal standard,73 which was not applicable in this case (for further information refer to section D.4). Nevertheless, a calibration was performed on a control kidney slice, which was spiked with different total amounts of a Pt standard solution, as has been

132 BAM-Dissertationsreihe Part D: Results and Discussion accomplished by Seuma et al. for the determination of Au in tissue sections.84 As illustrated in Fig. D.6.1.3-1, the 2D map shows four discrete locations of Pt where 500 (n=1), 50 (n=1), and 5 pg (n=2) of Pt were spiked on the control tissue slice. A calibration curve (y = 7 × 107x + 3 × 108) was established based on the 195Pt integrated areas of the Pt spot, after the substraction of integrated areas of 195Pt signal on unspiked blank tissue. Integration was performed by the software ImageJ. Application of the calibration to two replicates of the analyzed rat kidney tissue sections allowed the estimation of total Pt amounts in whole kidney slices. 35 and 42 ng Pt were calculated for the 16 mg kg-1 cisplatin slices, while 2 and 7 ng Pt were obtained for the 5 mg kg-1 cisplatin slices, showing the good reproducibility of the results. The limit of detection (3s of unspiked tissue) for Pt in a certain tissue volume (circle at 170 µm diameter in a 3 µm thin tissue section) was calculated to be as low as 50 fg. This value is in good agreement with solution-based LODs for Pt in the low pg g-1 range, assuming a 50 µL min-1 micronebulizer and a measurement time of 1 min. Considering the tissue slices weights, average contents of 14 and 144 µg Pt / g tissue were estimated for the 5 mg kg-1 cisplatin and the 16 mg kg-1 cisplatin kidneys, respectively. However, due to the inhomogeneous distribution of Pt in the tissue, the information given by a concentration regarding the whole thin section is rather limited. On the other hand, the total Pt amounts calculated for the 5 mg kg-1 cisplatin slices are lower than those for the 16 mg kg-1 cisplatin slices and hence, a correlation between drug dose and metal accumulation in the kidney slices is feasible. In the future, this quantification approach needs to be validated by digestion of a spiked tissue sections and analysis by ICP-MS with liquid sample introduction.

133 Part D: Results and Discussion

blank 5 pg 5 pg

50 pg 500 pg

170 µm

-1 Figure D.6.1.3-1 Control rat kidney tissue spiked with 0.5 µL of 1000, 100, and 10 ng g (n = 2) Pt standard solution. The 195Pt signals from LA-ICP-MS imaging of the spiked and unspiked areas marked in white and yellow, respectively, were integrated using ImageJ software. Figure adapted from reference 196, courtesy of Analytical Chemistry.

D.6.2 Copper and Zinc Bioimaging In addition to the investigation of Pt distribution in the rat kidneys presented in section D.6.1, Cu and Zn were simultaneously monitored to the Pt isotopes. The parallel, HE stained thin sections are given for comparison in Fig. D.6.2-1 g–i.

A control kidney section was analyzed by LA-ICP-MS to examine the copper and zinc distribution in a healthy, untreated rat. Copper accumulation in the cortex was observed, as illustrated in the corresponding image in Fig. D.6.2-1 a. This observation is underlined with the fact that copper transporters are located in the cell membranes of the cortical renal tubules.197 A slight decrease in the Cu signal, especially in the cortex, was detected upon 5 mg kg-1 bw and 16 mg kg-1 bw cisplatin treatment. The corresponding images are displayed in Fig. D.6.2-1 b, c. A copper decrease is in good agreement with previous observations198 and might be due to the fact that Cu and Pt transport is mediated by the same transporters.197 Thus, Cu might be replaced by Pt to some extent owing to competitive kinetics and their close atomic radii.199

On the other hand, Zn accumulation is slightly more located in the center of the tissue for the 5 mg kg-1 cisplatin-treated rat in comparison to the control sample (see Fig. D.6.2-1 d, e). In case of the 16 mg kg-1 cisplatin-treated rat a remarkably lower

134 BAM-Dissertationsreihe Part D: Results and Discussion signal for Zn is observed in the cortex, as illustrated in Fig. D.6.2-1 f. Instead, Zn is mainly accumulated in the medulla and papilla. Especially in the corticomedullary region where Pt was highly accumulated, Zn might be replaced by Pt. Although the Pt and Zn transporters are different, this observation corresponds to previous results.198 Furthermore, Zn and Pt have similar atomic radii,199 and the displacement of Zn by Pt has been reported in metallothioneins.200

65 66 Figure D.6.2-1 LA-ICP-MS images for the simultaneous monitoring of Cu (a–c), and Zn 195 (d–f). The corresponding Pt images are displayed in Fig. D.6.1.1-1 (f, e, a). (a, d, g): no -1 -1 cisplatin administration; (b, e, h): 5 mg kg cisplatin, (c, f, i): 16 mg kg cisplatin. (g–i): HE stained parallel sections. Figures adapted from reference 196, courtesy of Analytical Chemistry.

D.6.3 Platinum Bioimaging for the Evaluation of a Nephroprotector The bioimaging methodology described herein was also used to evaluate the protective effect of cilastatin on cisplatin toxicity in a rat model. Cilastatin is an inhibitor of renal dehydropeptidase I, located in the membrane of RPTECs, and

135 Part D: Results and Discussion reduces apical entry of cisplatin and other drugs into these cells.154, 194 Furthermore, the inhibitory effect of cilastatin was exclusively found in RPTECs and thus, is a promising nephroprotective agent, which might not alter cisplatin antitumor properties.194 Considering that cisplatin-induced renal damage and the related Pt accumulation take place mainly in the cortex, it is of particular interest to focus on this area during the study of nephroprotective strategies.

Comparison of Pt accumulation was made by LA-ICP-MS analysis in selected regions comprising the kidney cortex of rats treated with 5 mg kg-1 bw cisplatin alone, or combined with cilastatin administration. High spatial resolution images for 195Pt monitoring were obtained by performing adjacent line scans at 25 µm spot size, and 25 µm s-1 scan rate. Results are displayed in Fig. D.6.3-1 a, b, respectively. A considerable decrease in Pt signal was observed in the cortical area analyzed when cisplatin was co-administered with cilastatin (Fig. D.6.3-1 b), as compared to the cortex from the cisplatin-treated rat (Fig. D.6.3-1 a). Additionally, the HE stained sections of a kidney from a rat treated with cisplatin and cilastatin displayed in Fig. D.6.1.2-2 d exhibit less tubule damage and less protein casts than in the cisplatin-only treated rat (see Fig. D.6.1.2-2 b, c). These findings are in good correlation with previous studies, where the total Pt accumulation in the cortex upon cisplatin treatment was reduced about 22 % under the presence of cilastatin.201 Since Pt accumulation is mainly located in the cortex, no significant variations were observed in the medulla.201 As a result of the investigations on kidney tissue sections, cilastatin is a promising candidate for nephroprotective strategies during cisplatin treatments since it significantly reduced Pt accumulation in the cortex.

136 BAM-Dissertationsreihe Part D: Results and Discussion

195 Figure D.6.3-1 High spatial resolution LA-ICP-MS images for Pt monitoring on selected -1 cortical areas on sagittal kidney sections from rats treated with a 5 mg kg cisplatin, and b 5 mg kg-1 cisplatin + 150 mg kg-1 cilastatin per day (sacrifice after 5 days). Laser ablation parameters: 25 µm laser spot size, 25 µm s-1 scan speed, 20 Hz repetition rate, 33 % laser energy. Figures adapted from reference 196, courtesy of Analytical Chemistry.

D.6.4 Summary The effect of tissue dewaxing was investigated during this study. There was no Pt loss observed, but resolution in the paraffin-embedded sections seemed to be slightly

137 Part D: Results and Discussion lower, which is probably due to paraffin melting during the ablation process. Thus, dewaxed tissue sections were employed to achieve the highest attainable resolution to monitor Pt distribution in the main renal structures. The LA-ICP-MS-based Pt image of a whole rat kidney tissue was attained in an analysis time of 3 h with100 µm laser spot size, which is a rather short time for imaging a whole organ at high spatial resolution. The reproducibility of the method was shown by the determination of the total Pt amount in a tissue section, which was 35 and 42 ng for the 16 mg kg-1 treated rat, and 2 and 7 ng for the 5 mg kg-1 treated rat. The LOD of Pt in the tissue was estimated at 50 fg.

The method described herein permits the visualization of Pt distribution in kidney tissue at a microscopic scale, and hence provides important biological information on the toxicity of cisplatin. Application of 3 µm thin tissue sections resulted in high spatial resolution imaging preventing a mixing of different tissue layers. The distribution of Pt, Cu, and Zn was monitored in kidneys obtained from rats treated with zero, 5 mg kg-1, and 16 mg kg-1 cisplatin. Since Pt accumulates mainly in the cortex, renal damage occurred in this substructure. These findings were in good agreement with histomorphological studies of parallel HE stained thin sections. Moreover, a decrease of Cu and Zn was observed under cisplatin treatment, indicating the partial replacement of these metals by Pt. Furthermore, the nephroprotective effect of cilastatin upon cisplatin treatment was successfully investigated for the cortex region of a rat kidney.

The distribution of metals monitored by LA-ICP-MS in the kidney is changed by the treatment with cisplatin. This effect might be explained by the displacement of essential trace elements such as Cu and Zn by Pt, or by altered expression levels of metalloproteins. Additionally, the individual distribution patterns of the metals might reflect their different transport mechanisms in the kidney. In the future, these hypotheses need to be further investigated by identification of the proteins involved in these mechanisms. This can be accomplished by 2D-GE of protein extracts and subsequent nLC-ESI-MS/MS experiments, as has already been mentioned in section D.5.

138 BAM-Dissertationsreihe Part E: Summary and Outlook

E Summary and Outlook In this work, the successful development of ICP-MS-based assays for medical, biochemical, and environmental applications has been described. As model systems, the screening of tumor markers, metalloproteins, and metallodrugs has been accomplished. In order to establish a quantification of biomarkers in tissue sections, a suitable internal standard was investigated. Furthermore, LA-ICP-MS has been applied for single cell and cell nucleus imaging.

The analysis of biomolecules in a diversity of modifications requires the application and further development of highly sensitive detection methods, capable of tolerating a complex matrix. The search for molecular and genetic biomarkers can result in the detection of diseases at an early stage, or can guide treatment plans. However, the screening for biomarkers demands high throughput and multiplexing techniques considering the variety, and complexity of analytes to be queried. Finally, quantitative information on the expression level is highly valuable to identify abnormalities in a given organism.

To fulfil these requirements, the application of ICP-MS is promising due to its high sensitivity, wide linear dynamic range, and multielement capabilities. The main objective of this work was the development of sensitive techniques for the analysis of biomolecules via ICP-MS detection (i) of natural metal tags, or (ii) of chemical labels. Herein, four strategies were pursued:

1. The labeling via nanoparticles in ICP-MS-based assays;

2. The multiplexed detection of cancer biomarkers;

3. The use of iodine for the analysis of single cells, and its application as an internal standard for tissue samples;

4. The bioimaging of metalloproteins to study nephrotoxicity.

In the first section of this work, an immunoassay for OTA determination in wine was developed and tested with ICP-MS, and photometric detection. The detection limit of the assay was determined as 0.003 µg L-1, and the determination of OTA in wine was conducted with a quantification range of 0.01–1 µg L-1 by both detection

139 Part E: Summary and Outlook methods. In the future, other commodities such as coffee or cereals may be screened for OTA with the method presented here as well. Furthermore, the assay might be transferred to an array with subsequent detection by LA-ICP-MS, and targeted against different substances to increase sample throughput.

An array-based LA-ICP-MS approach for DNA microarray detection was elaborated in the second section of this work. In order to realize a high throughput screening, single pulse ablation of the spots was desirable, and has been accomplished with high signal intensities for the analyte of interest. However, the inhomogeneity of microarray spots hampered quantification, but might be improved by contactless printing of target oligonucleotides. Furthermore, the detection of single pulses in LA is still a challenge today due to the resulting short transient signals.202 A LA chamber providing optimum gas flows for these analyses is still under investigation.

The need for multiplex analyses in clinical diagnosis was met in the third section of this work. The analysis of subsequently stained tissue sections has been the gold standard in IHC so far, bearing the risk of low comparability owing to variant staining conditions. The application of LA-ICP-MS for IHC detection is advantageous since it provides the simultaneous monitoring of different parameters in a single experiment, and thus eliminating variability of the staining procedure. In this work, a multiplexed IHC was developed for up to three tumor markers in breast cancer tissue. In future work, the simultaneous detection of a higher number of biomarkers is of interest to further speed up diagnosis. Furthermore, the application of this technique to tissue microarrays has the potential to considerably increase sample throughput, since this technique allows the analysis of up to 1000 tissue samples on a single microscope glass slide.89 Up until now, resolution is still a factor of 10 to 100 inferior to light microscopy. By applying highly selective antibodies as well as highly amplifying labels, resolution in LA-ICP-MS might be increased to the cellular level, which is desirable to compete with routine IHC.

The high spatial resolution at a microscope scale was realized by iodine labeling of single cells and tissue samples in the fourth section of this work. The method resulted in elemental staining of the cell nuclei, which was represented by distinct iodine

140 BAM-Dissertationsreihe Part E: Summary and Outlook signals in LA-ICP-MS imaging at 4 µm laser spot size. Furthermore, iodine could be employed as an internal standard to correct for tissue thickness inhomogeneities.

In the fifth section of this work, the separation of proteins by one- and two-dimensional gel electrophoresis was elaborated for LA-ICP-MS detection to accomplish a pre-selection of platinated proteins from a cell lysate. The method developed herein enabled the identification of the protein spots of interest in a straightforward way. In the future, these protein spots may be excised and digested in a parallel experiment, with subsequent identification of proteins by nLC-ESI-MS/MS.

A methodology for the investigation of cisplatin-induced side-effects and of a nephroprotector was established in the sixth section of this work. LA-ICP-MS was employed for elemental imaging of kidney tissues from rats treated with cisplatin. The method developed herein has the potential to detect metals and metalloproteins in tissues at a microscopic scale. The application of this ‘elemental microscope’ enables a direct connection to histomorphological findings, and helps to gain a deeper insight into the underlying biochemical processes owing to the improved spatial resolution. The identification of metalloproteins in the tissue might be achieved by the complementary use of molecule specific MS in the future.

The advantages of ICP-MS for the life sciences lie in the potential of multiplexed, and sensitive quantitative analyses at high linear dynamic range. With this technique, a variety of modifications can be queried in a single experiment. However, there are a number of open questions to be solved:

1. The analysis of low abundant proteins still poses a challenge to ICP-MS detection, since only a few labels are conjugated with the analyte of interest. Hence, the amplification of the label either by converting it to a polymer tag, or by application of nanoparticles has been of interest recently.

2. The spatial resolution available for tissue imaging is still inferior to light microscopy. If amplifying labels were employed for analysis of tissue sections, spatial resolution might be reduced.

141 Part E: Summary and Outlook

3. The stoichiometry of labeled biomolecules is required for the quantification of biomolecules via chemical labels. Thus, more defined labeling strategies are advantageous.

4. Matrix-matched standards are needed for quantification. They might be produced by defined labeling strategies.

Considering the use of nanoparticles, new multiplexing strategies have to be developed, which are comparable to the possibilities provided by lanthanide labels. This problem could be solved by the combination of different elements in a nanoparticle at known mass ratios, which might be applied for single cell analysis. A separation of the biomolecules of interest is a prerequisite to sustain the characteristic elemental ratios in this case. On the other hand, the use of polymertags is more promising for a multiplexed IHC analysis of co-localized tumor markers. However, nonspecific binding on the sample surface lowers signal-to-background ratios and thus, impedes the application of these signal-amplifying tags unless suitable blocking conditions are elaborated.

For the quantification of biomolecules by ICP-MS, more defined labeling strategies are needed. This might be achieved by means of biotechnology. A specific amino acid sequence in a protein’s primary structure could be recognized by an enzyme, which then attaches a label.203 The defined labeling of standard proteins might be useful to design matrix-matched standards. For nanoparticle tagging, quantification might be facilitated by nanoparticle calibration, based on the work by Garcia et al.204

To conclude, ICP-MS is capable of the detection and quantification of proteins or protein modifications, but other mass spectrometry techniques like ESI or MALDI are needed for their identification. Consequently, the complementary application of elemental and molecule specific mass spectrometry techniques is highly desirable to tackle complex questions. The identification of new biomarkers is essential for personalized medicine, but reliable biomarkers have not been identified yet for a number of diseases. This can only be accomplished by a multidisciplinary approach involving proteomics-based technologies, and computational modeling.205

142 BAM-Dissertationsreihe Part F: Appendix

F Appendix

F.1 Nano Electrospray Ionization Time-of-Flight Mass Spectrometry (nESI-TOF-MS) The term ‘electrospray’ describes the generation of droplets through application of a high voltage to a liquid flow at atmospheric pressure.206, 207 Electrospray ionization (ESI) is a soft ionization method, which is capable of producing ionized macromolecules.6 The nano-electrospray technique was introduced in 1994208, 209 and refers to flow rates of about 10–20 nL min-1.111 In offline mode, the sample is loaded into metal coated, disposable capillaries. The flow starts when voltage is applied and continues until the sample is depleted. Owing to the voltage on the capillary, the sample spray is charged as it is nebulized. The charged droplets evaporate in the interface, and as the solvent is removed, the droplets shrink at constant charge. At a certain point, droplets disintegrate due to Coulomb-explosion into droplets of a few nm size, and finally to single analyte ions.

From the interface, ions are transferred to a mass spectrometer. In this work, a time-of-flight (TOF) instrument (Q-TOF 2, MS Vision, Goch, Germany) was used. This instrument is especially suited for the analysis of high mass biomolecules, since they are slowed by a high-pressure (in this case 25 mbar) in front of the first hexapole. This technique induces collisional cooling and focusing of large ions.210 In the quadrupole, selected ions are transmitted, and subsequently enter the TOF mass analyzer. This technique is based on the measurement of the flight time of ions with known energy over a known distance. The ion beam is accelerated to a constant kinetic energy (zV), where z is the charge on the ion and V is the applied accelerating potential. The ions enter a drift tube and acquire a characteristic velocity dependent on their m / z:

m / z = 2Vt² / l², where t is the flight time, and l is the flight path length. Thus, the time of flight is proportional to 1 / V1/2. Ions of different mass travel down the drift tube at different velocities. Therefore, light ions which are traveling faster reach the detector at the end of the drift tube in a shorter time than heavier ions. This time discrimination

143 Part F: Appendix process can be used to resolve ions of different mass. Resolution depends on having an ion source that produces ions of a very low initial energy spread. Improvements in resolution have been accomplished with the use of reflection devices. After traveling through the field-free region, as in the simple linear TOF, ions are subjected to an electrostatic field that serves to decelerate and reflect them. Resolution is improved because slower ions of the same mass are allowed to "catch up" with faster ones by spending less time in the reflector region. In any TOF instrument, the ion source must be operated in a pulsed mode to define the starting point for the measurement of the flight time. The major advantage of TOF-MS over other forms of mass spectrometry is the ability to monitor the m / z of all the ions simultaneously.

F.2 Analysis of MUC 1 (Tb) by nESI-Q-TOF-MS The antibodies previously investigated carried an average of two DOTA labels.57 Since MUC 1 (Tb) exhibited more than two orders of magnitude higher sensitivities than Her 2 (Ho) or CK 7 (Tm) in the multiplexed IHC experiment presented in section D.3, its labeling degree was verified by nESI-Q-TOF-MS. A borosilicate needle, coated with Au / Pd (Proxeon, Thermo Fisher Scientific, Dreieich, Germany) was used for nESI, and a 1 kV capillary voltage was applied for ionization. The antibody was suspended in 200 mM ammonium acetate buffer pH 7 and hence, the charge carriers were ammonium ions (mass: 18 Da).

Figures F.2-1 and F.2-2 illustrate that m / z decreased by the increase of collision energy, which caused loss of solvent molecules. Hence, the exact mass of the anti-MUC 1 was determined as 147779 Da (no. 0). The peaks of the DOTA (Tb) labeled anti-MUC 1 (MUC 1 (Tb)) were split into three (see Fig. F.2-1), and the corresponding masses were 149172 Da (no. 3), 148508 Da (no. 2), and 147782 Da (no. 1). The mass difference 2–0 was 726 Da, and 3–0 was 1390 Da, taking 1–0 (3 Da) as basic shift. Thus, mass no. 1, 2, and 3 represented the conjugate with a complete loss of label, with one DOTA label, and with two DOTA labels, respectively as specified in Table F.2-1. The mass differences 2–3 (665 Da) and 1–2 (726 Da) represented the loss of one label, whereas 1–3 (1390 Da) indicated the loss of two labels. The label fragments, which might explain the calculated mass differences, are displayed in Fig. F.2-3. Hence, the determined mass of non-labeled antibody correspond with the findings of the labeled antibody assuming an average labeling of

144 BAM-Dissertationsreihe Part F: Appendix two per anti-MUC 1, which is in good agreement with previous studies.57 Furthermore, a labeling degree of one, and non-conjugated antibody were detected. As a result, these investigations indicate that the higher sensitivities in LA-ICP-MS for Tb were caused by a higher expression level of MUC 1 compared to Her 2 or CK 7.

Table F.2-1 Mass differences between labeled and non-labeled anti-MUC 1. 0: non-labeled antibody, 1: complete loss of label, 2: one label, 3: two labels.

Mass difference [Da] Corrected mass difference [Da] Labels

1-0 3 0 2-0 729 726 1 3-0 1393 1390 2

1-2 726 1 2-3 665 1 1-3 1390 2

145 Part F: Appendix

Figure F.2-1: Analysis of MUC 1 (Tb) by nESI-Q-TOF-MS, increase of collision energy up to 140 eV.

Figure F.2-2 Analysis of non-labeled MUC 1 (green line), and MUC 1 (Tb) (blue line) by nESI-Q-TOF-MS, increase of collision energy up to 100 eV (green line), and up to 140 eV (blue line).

146 BAM-Dissertationsreihe Part F: Appendix

CO 2 CO2

N N

H3N CO2

N +3 N Tb N Tb+3 N

CO2

N N

HO C HO2C 2

Exact Mass: 664,12 Exact Mass: 695,17 Molecular Weight: 664,51 Mol. Wt.: 695,57 NCS NCS

CO2

NH4 N

O2C

N Tb+3 N

CO2 N

HO2C

Exact Mass: 725,14 Mol. Wt.: 725,55

NCS

Figure F.2-3 Structues, which might explain the mass differences in Table F-1 (665 Da, 726 Da, and 1390 Da (= 2 × 695 Da).

F.3 Application of Highly Amplifiying Labels to LA-ICP-MS-based IHC Currently, resolution of LA-ICP-MS-based IHC is still a factor of 10 to 100 inferior to light microscopy. Thus, highly amplifying labels based on polymer tags46 or nanoparticle tags were employed for IHC.

147 Part F: Appendix

The tested polymertags were purchased from MaxPar (DVS Sciences, Canada). These labels were developed for signal amplification in liquid ICP-MS. For LA-ICP-MS-based IHC analysis, MUC 1 was labeled with Tm loaded polymertag according to the manufacturer’s instructions, and applied to a 5 µm breast cancer tissue at a concentration of 1 µg mL-1 (the same concentration as DOTA labeled MUC 1) for one hour (a shorter incubation compared to DOTA labeled MUC 1, which was incubated for three hours). The results, which are displayed in Fig. F.3-1, demonstrate a significant nonspecific binding of the polymer to the sample surface, which caused up to 1 × 106 cps for the negative control (palatine tonsil tissue). The positive control (breast cancer tissue) yields 1 × 107 cps and thus, the signal-to-background ratio was approximately 10. By further decreasing the antibody concentration to 10 ng mL-1, and a reduction of the incubation time to 30 minutes, the signal-to-background ratio was approximately 4. Hence, these results demonstrate the difficulty in applying polymertags to LA-ICP-MS-based IHC. To conclude, the application of polymertags to thin tissue sections, combined with LA-ICP-MS is not straightforward due to nonspecific binding to the sample surface and needs further optimization.

148 BAM-Dissertationsreihe Part F: Appendix

Figure F.3-1 Single line scan by LA-ICP-MS of a 5 µm tissue sections, on which MUC 1 labeled with Tm loaded polymertag was applied at a concentration of 1 µg mL-1 for one hour was. Laser parameters: 200 µm laser spot size, 200 µm s-1 scan speed, 20 Hz repetition rate, and 35 % laser energy. a breast cancer tissue (positive control), b palatine tonsil tissue (negative control).

On the other hand, gold nanoparticle labels were applied to LA-ICP-MS-based IHC as well via a conjugate with secondary antibodies (gold labeled anti-mouse IgG (H+L) System 40 nm, goat, 0.1 mg mL-1 (lot 090028) from KPL (Gaithersburg, MD, USA)). For this purpose, a thyroid gland tissue was sectioned at 5 µm thickness setting, and incubated with 31 µg mL-1 TTF 1 (anti-thyroid transcription factor, Dako Deutschland GmbH, Hamburg, Germany) for one hour. The tissue was blocked for one hour with 0.1 % BSA in PBS buffer prior to incubation with the secondary antibody. Finally, the tissue was incubated with a secondary antibody concentration of 1 µg mL-1 for 30 minutes.

149 Part F: Appendix

Figure F.3-2 LA-ICP-MS images of 5 µm thyroid gland tissue, incubated with TTF 1 and gold 127 197 nanoparticle labeled secondary antibodies. a I signal, b Au signal. Laser parameters: Laser spot size 100 µm, scan speed 90 µm s-1, repetition rate 5 Hz, and laser energy 35 %.

For LA-ICP-MS measurements, the morphology of the thyroid glands in the tissue was monitored by detection of 127I, and the gold nanoparticle labels were monitored by 197Au. As illustrated in Fig. F.3-2 a, iodine was detected with signal intensities of up to 3 × 107 cps, resulting in a good visualization of individual thyroid glands. On the contrary, the Au signal in Fig. F.3-2 b was distributed throughout the whole tissue sections, albeit TTF 1 only binds to cell nuclei of the thyroid glands according to conventional IHC findings (not shown here). Thus, the binding of gold nanoparticle labels was nonspecific in this case, albeit a blocking step with BSA had been performed on the tissue prior to labeling. The results demonstrated again the difficulty in applying highly amplifying labels to LA-ICP-MS-based IHC. In the future, the enhancement of signal-to-background ratios might be achieved by optimizing the blocking conditions, as well as the applied antibody concentrations.

150 BAM-Dissertationsreihe Part F: Appendix

F.4 Gold Nanoparticle Incubation of Fibroblast Cells and Detection by LA-ICP-MS It is known from previous observations by Kneipp et al. that living cells can take up gold nanoparticles to the cytoplasma.211 For the analysis of fibroblasts by LA-ICP-MS, cells were incubated with 30 nm gold nanoparticle at a particle concentration of 100 pM for six hours. Cells were fixed according to the protocol presented in section C.4, and subsequently ablated by adjacent line scans at 4 µm laser spot size, and 4 µm s-1 scan speed. The corresponding LA-ICP-MS image of the 197Au intensity distribution is shown in Fig. F.4-1, along with a microscope photograph of the cells prior to ablation. Gold intensities were measured in the cytoplasm at intensities of up to 106 cps, whereas gold signal was absent in the cell nuclei, which is a hint that these findings are in good agreement with previous observations.211 Furthermore, 30 nm gold nanoparticles contain approximately 2 × 106 gold atoms and hence, these high signal intensities can only be explained by the formation and detection of gold nanoparticle aggregates. However, in addition to cell nuceus imaging by iodine labeling, the uptake of nanoparticles to the cytoplasm can be monitored by LA-ICP-MS imaging of fibroblast cells with single cell resolution, enabling studies on metal nanoparticle toxicity and transport in cells.

151 Part F: Appendix

Figure F.4-1 Photograph taken prior to laser ablation (lower part), and 197Au intensity surface plot of fibroblast cells (upper part), which were incubated with 30 nm Au nanoparticles. Laser parameters: laser spot size 4 µm, scan speed 4 µm s-1, repetition rate 20 Hz, laser energy 45 %.

F.5 Imaging of Thyroid Gland Tissue by LA-ICP-MS Naturally abundant iodine is found in thyroid glands, and can also be monitored by LA-ICP-MS as illustrated in Fig. F.3-2 a. In this experiment, signal intensities for 127I were as high as 3 × 107 cps. Thus, the spatial resolution can be further enhanced. For this purpose, a human thyroid gland tissue slice was sectioned with a microtome at a thickness setting of 3 µm, and analyzed by LA-ICP-MS at a laser spot size of 12 µm. A photograph of the ablated tissue area was taken prior to laser ablation and is displayed in Fig. F.5-1. The tissue was ablated line by line, and the corresponding surface plot of 127I distribution yielded intensities of up to 105 cps. Characteristic morphological features of the thin section were in good agreement with the microscope photograph presented in Fig. D.4.6-1 (lower part).

152 BAM-Dissertationsreihe Part F: Appendix

Moreover, the LA-ICP-MS image holds information on the relative iodine abundance within each individual thyroid gland. For thyroid gland tissue analysis, this method can be employed to monitor up-regulated or down-regulated iodine metabolism, in addition to IHC analyses applying anti-thyroglobulin, which is also used to monitor iodine metabolism in thyroid glands.212

Figure F.5-1 Human thyroid gland. Photograph taken prior to laser ablation (lower part), and LA-ICP-MS image of 127I distribution (upper part). Laser parameters: laser spot size 12 µm, scan speed 10 µm s-1, repetition rate 5 Hz, laser energy 35 %.

153

Acknowledgments

Acknowledgments I am indebted to my many colleagues and friends for their support, lab assistance, and our fruitful discussions over the years. The research work I was able to perform at BAM has been inspiring and encouraging. Thank you for making this work possible.

I am deeply grateful to my PhD supervisor Ulrich Panne for his ongoing support and encouragement. The confidence he has in me as a person and in my work has given me freedom in my research work and hence, personal fulfillment.

I would like to express my gratitude to Norbert Jakubowski and to Michael G. Weller. Their outstanding support and countless scientific discussions were invaluable to me for the successful completion of this PhD thesis.

I am indebted to Thomas Mairinger from HELIOS Klinikum Emil von Behring. His optimism and encouragement has given me the chance to start our successful collaboration in immunohistochemistry and histology.

This work would not have been possible without the support of many colleagues. My sincere thanks to Heike Traub for the introduction to laser ablation and for numerous discussions at any time of the day; Larissa Wäntig for antibody labeling advice; Rajko Winkler for skillful performance of the nESI-Q-TOF-MS analysis of anti-MUC 1 at Linscheid Lab (HU Berlin); Daniela Drescher and Janina Kneipp for the cooperation regarding fibroblast cells; the working group of Heinrich Kipphardt for support, and especially Andreas Schulz for technical advice in ICP-MS; Norbert Jakubowski for thoroughly reviewing my papers; Julia Wienold and Silke Richter for helpful discussions; Jens Riedel for an introduction to ImageJ; Wilfried Weigel for advice in microarray handling and readout by a microarray scanner at Scienion AG; Franziska Emmerling for SAXS measurements of the Streptavidin-Au conjugate to verify the nanoparticle size stated by the manufacturer; Manfred Hirsch and Christoph Naese for skillful manufacturing of a laser ablation chamber bracket, and a sample holder; Ute Resch-Genger and Thomas Behnke for testing Eu-particles on tissue sections; Uwe Reinholz for testing TXRF measurements of microarray samples; Silke Richter

155 Acknowledgments for testing ETV-ICP-MS measurements of Au particles; Katrin Hoffmann for testing fluorescence microscopy of microarray samples.

Lina Khoury (HELIOS Klinikum Emil von Behring) is greatly acknowledged for introducing me to immunohistochemistry, for her lab assistance in handling thin tissue sections, and HE staining.

I would like to express my gratitude to Estefanía Moreno-Gordaliza, Diego Esteban- Fernández, and M. Milagros Gómez-Gómez for our outstandingly fruitful collaboration to study cisplatin-induced nephrotoxicity. My sincere thanks to Karola Lehmann and Christian Scheler from Proteome Factory AG for collaboration and advice regarding 2D gel electrophoresis, which was essential for the project on platinated proteins performed with Estefanía Moreno-Gordaliza.

I am deeply grateful to Detlef Günther for his encouragement and support, and for giving me the opportunity to finally analyze the microarray samples by LA-ICP-MS at ETH Zürich, as well as officiating as my third referee. My sincere thanks to Mattias Fricker for planning and conducting the microarray experiments with me.

I would like to thank Rudolf Schneider and Michael G. Weller for research advice regarding ELISA, and Sabine Flemig and Kristin Petsch for technical advice. Skadi Kull is gratefully acknowledged for assistance in assay optimization.

Juliane Schaefer and Christin Heinrich are gratefully acknowledged for facilitating everyday life, and Anka Kohl for her skillful IT support.

I am indebted to Michael W. Linscheid, who officiated as my second referee, and for giving me the opportunity to engage in interesting collaborations.

I would like to express my gratitude to Julia Wienold and Norbert Jakubowski, who thoroughly read the manuscript and suggested improvements, to Mattias Fricker who commented on the microarray section, and to Ulrike Hochkirch, who read the nESI section. Eugene Milstone, my dear friend, and his son David Milstone are greatly acknowledged for reviewing the English language.

My sincere thanks to my PhD colleagues Virginia Joseph, Andrea Matschulat, Jonas Schenk, Astrid Walter, and Julia Grandke for sharing daily challenges.

156 BAM-Dissertationsreihe Acknowledgments

Last but not least I would like to thank my family and friends, whose support directly influenced the success of my work. A special thank to my parents who gave me the opportunity to start a career in science. Many ideas arose during discussions with friends, and I thank Stefanie Markowetz, Eugene Milstone, Larissa Wäntig, Estefanía Moreno-Gordaliza, and friends at the Western Stable Werneuchen. I am deeply grateful to Nicole Seidel & Nicole Risse for their beautiful cover design for the JAAS Issue 11 in 2011, and for the cover of my thesis.

I am particularly indebted to my wife Jana Giesen, since this work would have been impossible for me to finish without her everlasting support. Many thanks to her for Chyara Skip, who has taught me self-confidence, assertiveness, and self-reflection.

Yours sincerely,

Charlotte Giesen

157

Literature

Literature 1. Consortium, International Human Genome Sequencing, Finishing the euchromatic sequence of the human genome. Nature 2004, 431, 931-945. 2. van Ommen, G. J. B., The human genome, revisited. European Journal of Human Genetics 2005, 13 (3), 265-267. 3. van Ommen, G. J. B., The Human Genome Project and the future of diagnostics, treatment and prevention. Journal of Inherited Metabolic Disease 2002, 25 (3), 183-188. 4. Lottspeich, F.; Engels, J. W., Bioanalytik. 2nd ed.; Elsevier GmbH: 2006. 5. Patterson, S. D.; Aebersold, R. H., Proteomics: the first decade and beyond. Nature Genetics 2003, 33, 311-323. 6. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization for Mass-Spectrometry of Large Biomolecules. Science 1989, 246 (4926), 64-71. 7. Karas, M.; Hillenkamp, F., Laser Desorption Ionization of Proteins with Molecular Masses Exceeding 10000 Daltons. Analytical Chemistry 1988, 60 (20), 2299-2301. 8. Yalow, R. S.; Berson, S. A., Radioimmunoassasy of gastrin. Gastroenterology 1970, 58 (1), 1-14. 9. Engvall, E.; Perlman, P., Enzyme-linked immunosorbent assay (ELISA) Quantitative assay of immunoglobulin G. Immunochemistry 1971, 8 (9), 871-874. 10. Ekins, R. P., ELISA - A replacement for radioimmunoassays? The Lancet 1976, 308 (7982), 406-407. 11. Avrameas, S., Enzyme-Linked Immunosorbent Assay, Elisa: III. Quantitation of Specific Antibodies by Enzyme-Labeled Anti-Immunoglobulin in Antigen-Coated Tubes. International Review of Cytology 1970, 27, 349-385. 12. Rüdiger, T.; Höfler, H.; Kreipe, H. H.; Nizze, H.; Pfeifer, U.; Stein, H.; Dallenbach, F. E.; Fischer, H. P.; Mengel, M.; von Wasielewski, R.; Müller-Hermelink, H. K., Interlaboratory trial 2000 "Immunohistochemistry" of the German Society for Pathology and the Professional Association of German Pathologists. Pathologe 2003, 24 (1), 70-78.

159 Literature

13. Scheele, G. A., 2-Dimensional Gel Analysis of Soluble-Proteins - Characterization of Guinea-Pig Exocrine Pancreatic Proteins. Journal of Biological Chemistry 1975, 250 (14), 5375-5385. 14. Klose, J., Protein Mapping by Combined Isoelectric Focusing and Electrophoresis of Mouse Tissues - Novel Approach to Testing for Induced Point Mutations in Mammals. Humangenetik 1975, 26 (3), 231-243. 15. Zhang, H.; Yan, W.; Aebersold, R., Chemical probes and tandem mass spectrometry: a strategy for the quantitative analysis of proteomes and subproteomes. Current Opinion in Chemical Biology 2004, 8 (1), 66-75. 16. Ong, S. E.; Blagoev, B.; Kratchmarova, I.; Kristensen, D. B.; Steen, H.; Pandey, A.; Mann, M., Stable isotope labeling by amino acids in cell culture, SILAC, as a simple and accurate approach to expression proteomics. Molecular & Cellular Proteomics 2002, 1 (5), 376-386. 17. Gygi, S. P.; Rist, B.; Gerber, S. A.; Turecek, F.; Gelb, M. H.; Aebersold, R., Quantitative analysis of complex protein mixtures using isotope-coded affinity tags. Nature Biotechnology 1999, 17 (10), 994-999. 18. Ross, P. L.; Huang, Y. L. N.; Marchese, J. N.; Williamson, B.; Parker, K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.; Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.; Jacobson, A.; Pappin, D. J., Multiplexed protein quantitation in Saccharomyces cerevisiae using amine-reactive isobaric tagging reagents. Molecular & Cellular Proteomics 2004, 3 (12), 1154-1169. 19. Gerber, S. A.; Rush, J.; Stemman, O.; Kirschner, M. W.; Gygi, S. P., Absolute quantification of proteins and phosphoproteins from cell lysates by tandem MS. Proceedings of the National Academy of Sciences of the United States of America 2003, 100 (12), 6940-6945. 20. Linscheid, M. W., Quantitative proteomics. Analytical and Bioanalytical Chemistry 2005, 381 (1), 64-66. 21. Prange, A.; Proefrock, D., Chemical labels and natural element tags for the quantitative analysis of bio-molecules. Journal of Analytical Atomic Spectrometry 2008, 23 (4), 432-459. 22. Thomas, R., Practical Guide to ICP-MS, A Tutorial for Beginners. 2nd ed.; Taylor Francis Group: Boca Raton, FL, U.S.A., 2008.

160 BAM-Dissertationsreihe Literature

23. Baranov, V. I.; Quinn, Z.; Bandura, D. R.; Tanner, S. D., A sensitive and quantitative element-tagged immunoassay with ICPMS detection. Analytical Chemistry 2002, 74 (7), 1629-1636. 24. Becker, J. S.; Jakubowski, N., The synergy of elemental and biomolecular mass spectrometry: new analytical strategies in life sciences. Chemical Society Reviews 2009, 38 (7), 1969-1983. 25. Bandura, D. R.; Baranov, V. I.; Ornatsky, O. I.; Antonov, A.; Kinach, R.; Lou, X. D.; Pavlov, S.; Vorobiev, S.; Dick, J. E.; Tanner, S. D., Mass Cytometry: Technique for Real Time Single Cell Multitarget Immunoassay Based on Inductively Coupled Plasma Time-of-Flight Mass Spectrometry. Analytical Chemistry 2009, 81 (16), 6813-6822. 26. Rosenzweig, A. C., Metallochaperones: Bind and deliver. Chemistry & Biology 2002, 9 (6), 673-677. 27. Pisonero, J.; Fernandez, B.; Günther, D., Critical revision of GD-MS, LA-ICP- MS and SIMS as inorganic mass spectrometric techniques for direct solid analysis. Journal of Analytical Atomic Spectrometry 2009, 24 (9), 1145-1160. 28. Qin, Z. Y.; Caruso, J. A.; Lai, B.; Matusch, A.; Becker, J. S., Trace metal imaging with high spatial resolution: Applications in biomedicine. Metallomics 2011, 3 (1), 28-37. 29. Zhang, C.; Wu, F. B.; Zhang, Y. Y.; Wang, X.; Zhang, X. R., A novel combination of immunoreaction and ICP-MS as a hyphenated technique for the determination of thyroid-stimulating hormone (TSH) in human serum. Journal of Analytical Atomic Spectrometry 2001, 16 (12), 1393-1396. 30. Zhang, C.; Wu, F. B.; Zhang, X. R., ICP-MS-based competitive immunoassay for the determination of total thyroxin in human serum. Journal of Analytical Atomic Spectrometry 2002, 17 (10), 1304-1307. 31. Karapitta, C. D.; Xenakis, A.; Papadimitriou, A.; Sotiroudis, T. G., A new homogeneous enzyme immunoassay for thyroxine using glycogen phosphorylase b- thyroxine conjugates. Clinica Chimica Acta 2001, 308 (1-2), 99-106. 32. Baranov, V. I.; Quinn, Z. A.; Bandura, D. R.; Tanner, S. D., The potential for elemental analysis in biotechnology. Journal of Analytical Atomic Spectrometry 2002, 17 (9), 1148-1152.

161 Literature

33. Zhang, C.; Zhang, Z. Y.; Yu, B. B.; Shi, J. J.; Zhang, X. R., Application of the biological conjugate between antibody and colloid Au nanoparticles as analyte to inductively coupled plasma mass spectrometry. Analytical Chemistry 2002, 74 (1), 96-99. 34. Quinn, Z. A.; Baranov, V. I.; Tanner, S. D.; Wrana, J. L., Simultaneous determination of proteins using an element-tagged immunoassay coupled with ICP- MS detection. Journal of Analytical Atomic Spectrometry 2002, 17 (8), 892-896. 35. Ornatsky, O. I.; Baranov, V.; Bandura, D.; Tanner, S.; Dick, J., Multiplex biomarker detection by ICP-MS. Faseb Journal 2006, 20 (4), A100-A100. 36. Ornatsky O.; Baranov V.; Bandura D.R.; Tanner S.D.; J., D., Messenger RNA Detection in Leukemia Cell lines by Novel Metal-Tagged in situ Hybridization using Inductively Coupled Plasma Mass Spectrometry. Translational Oncogenomics 2006, 1, 1-9. 37. Careri, M.; Elviri, L.; Mangia, A.; Mucchino, C., ICP-MS as a novel detection system for quantitative element-tagged immunoassay of hidden peanut allergens in foods. Analytical and Bioanalytical Chemistry 2007, 387 (5), 1851-1854. 38. Whetstone, P. A.; Butlin, N. G.; Corneillie, T. M.; Meares, C. F., Element- coded affinity tags for peptides and proteins. Bioconjugate Chemistry 2004, 15 (1), 3- 6. 39. Bunzli, J. C. G., Benefiting from the unique properties of lanthanide ions. Accounts of Chemical Research 2006, 39 (1), 53-61. 40. Waentig, L. Strategien für das Multielement-Labeling von Proteinen und Antikörpern für LA-ICP-MS-Anwendungen in der Proteomik. Technische Universität Dortmund, Dortmund, 2010. 41. Terenghi, M.; Elviri, L.; Careri, M.; Mangia, A.; Lobinski, R., Multiplexed Determination of Protein Biomarkers Using Metal-Tagged Antibodies and Size Exclusion Chromatography-Inductively Coupled Plasma Mass Spectrometry. Analytical Chemistry 2009, 81 (22), 9440-9448. 42. Krause, M., Scheler, C., Böttger, U., Weisshoff, H., Linscheid, M. W. Verfahren und Reagenz zur spezifischen Identifizierung und Quantifizierung von einem oder mehreren Proteinen in einer Probe. 10227599 B4, 2005. 43. Ahrends, R.; Pieper, S.; Kühn, A.; Weisshoff, H.; Hamester, M.; Lindemann, T.; Scheler, C.; Lehmann, K.; Taubner, K.; and Linscheid, M. W., A metal-coded

162 BAM-Dissertationsreihe Literature affinity tag approach to quantitative proteomics. Molecular & Cellular Proteomics 2007, 6 (11), 1907-1916. 44. Linscheid, M. W.; Ahrends, R.; Pieper, S.; Neumann, B.; Scheler, C., Metal- Coded Affinity Tag Labeling: A Demonstration of Analytical Robustness and Suitability for Biological Applications. Analytical Chemistry 2009, 81 (6), 2176-2184. 45. Esteban-Fernández, D.; Scheler, C.; Linscheid, M. W., Absolute protein quantification by LC-ICP-MS using MeCAT peptide labeling. Analytical and Bioanalytical Chemistry 2011, 401 (2), 657-666. 46. Lou, X. D.; Zhang, G. H.; Herrera, I.; Kinach, R.; Ornatsky, O.; Baranov, V.; Nitz, M.; Winnik, M. A., Polymer-based elemental tags for sensitive Bioassays. Angewandte Chemie-International Edition 2007, 46 (32), 6111-6114. 47. Bendall, S. C.; Simonds, E. F.; Qiu, P.; Amir, E. A. D.; Krutzik, P. O.; Finck, R.; Bruggner, R. V.; Melamed, R.; Trejo, A.; Ornatsky, O. I.; Balderas, R. S.; Plevritis, S. K.; Sachs, K.; Pe'er, D.; Tanner, S. D.; Nolan, G. P., Single-Cell Mass Cytometry of Differential Immune and Drug Responses Across a Human Hematopoietic Continuum. Science 2011, 332 (6030), 687-696. 48. Winnik, M. A.; Abderahman, A. I.; Thickett, S. C.; Liang, Y.; Ornatsky, O.; Baranov, V., Surface Functionalization Methods To Enhance Bioconjugation in Metal- Labeled Polystyrene Particles. Macromolecules 2011, 44 (12), 4801-4813. 49. Marshall, P.; Heudi, O.; Bains, S.; Freeman, H. N.; Abou-Shakra, F.; Reardon, K., The determination of protein phosphorylation on electrophoresis gel blots by laser ablation inductively coupled plasma-mass spectrometry. Analyst 2002, 127 (4), 459- 461. 50. Wind, M.; Feldmann, I.; Jakubowski, N.; Lehmann, W. D., Spotting and quantification of phosphoproteins purified by gel electrophoresis and laser ablation- element mass spectrometry with phosphorus-31 detection. Electrophoresis 2003, 24 (7-8), 1276-1280. 51. Chéry, C. C.; Günther, D.; Cornelis, R.; Vanhaecke, F.; Moens, L., Detection of metals in proteins by means of polyacrylamide gel electrophoresis and laser ablation-inductively coupled plasma-mass spectrometry: Application to selenium. Electrophoresis 2003, 24 (19-20), 3305-3313.

163 Literature

52. Ma, R. L.; McLeod, C. W.; Tomlinson, K.; Poole, R. K., Speciation of protein- bound trace elements by gel electrophoresis and atomic spectrometry. Electrophoresis 2004, 25 (15), 2469-2477. 53. Stumpf, S. Imaging von Proteinen durch Elementmarkierung mittels LA-ICP- MS. Humboldt Universität zu Berlin, Berlin, 2011. 54. Miller, I.; Crawford, J.; Gianazza, E., Protein stains for proteornic applications: Which, when, why? Proteomics 2006, 6 (20), 5385-5408. 55. Jiménez, M. S.; Rodriguez, L.; Gomez, M. T.; Castillo, J. R., Metal-protein binding losses in proteomic studies by PAGE-LA-ICP-MS. Talanta 2010, 81 (1-2), 241-247. 56. Müller, S. D.; Diaz-Bone, R. A.; Felix, J.; Goedecke, W., Detection of specific proteins by laser ablation inductively coupled plasma mass spectrometry (LA-ICP- MS) using gold cluster labelled antibodies. Journal of Analytical Atomic Spectrometry 2005, 20 (9), 907-911. 57. Waentig, L.; Roos, P. H.; Jakubowski, N., Labelling of antibodies and detection by laser ablation inductively coupled plasma mass spectrometry. Journal of Analytical Atomic Spectrometry 2009, 24 (7), 924-933. 58. Hu, S. H.; Zhang, S. C.; Hu, Z. C.; Xing, Z.; Zhang, X. R., Detection of multiple proteins on one spot by laser ablation inductively coupled plasma mass spectrometry and application to immuno-microarray with element-tagged antibodies. Analytical Chemistry 2007, 79 (3), 923-929. 59. Jakubowski, N.; Messerschmidt, J.; Anorbe, M. G.; Waentig, L.; Hayen, H.; Roos, P. H., Labelling of proteins by use of iodination and detection by ICP-MS. Journal of Analytical Atomic Spectrometry 2008, 23 (11), 1487-1496. 60. Waentig, L.; Jakubowski, N.; Hayen, H.; Roos, P. H., Iodination of proteins, proteomes and antibodies with potassium triodide for LA-ICP-MS based proteomic analyses. Journal of Analytical Atomic Spectrometry 2011, 26 (8), 1610-1618. 61. Hunter, W. M.; Greenwood, F. C., Preparation of Iodine-131 Labelled Human Growth Hormone of High Specific Activity. Nature 1962, 194 (4827), 495-&. 62. Fraker, P. J.; Speck, J. C., Protein and Cell-Membrane Iodinations with a Sparingly Soluble Chloramide, 1,3,4,6-Tetrachloro-3a,6a-Diphenylglycoluril. Biochemical and Biophysical Research Communications 1978, 80 (4), 849-857.

164 BAM-Dissertationsreihe Literature

63. Espuña, G.; Andreu, D.; Barluenga, J.; Pérez, X.; Planas, A.; Arsequell, G.; Valencia, G., Iodination of proteins by IPy2BF4, a new tool in protein chemistry. Biochemistry 2006, 45 (19), 5957-5963. 64. Bolton, A. E.; Hunter, W. M., Labeling of Proteins to High Specific Radioactivities by Conjugation to a I-125-Containing Acylating Agent - Application to Radioimmunoassay. Biochemical Journal 1973, 133 (3), 529-538. 65. Vaidyanathan, G.; Zalutsky, M. R., Preparation of N-succinimidyl 3- [*I]iodobenzoate: an agent for the indirect radioiodination of proteins. Nature Protocols 2006, 1 (2), 707-713. 66. Fletcher, C. D. M., Diagnostic Histopathology of Tumors. 3rd ed.; Churchill Livingstone: 2007. 67. Becker, J. S.; Matusch, A.; Palm, C.; Salber, D.; Morton, K. A.; Becker, S., Bioimaging of metals in brain tissue by laser ablation inductively coupled plasma mass spectrometry (LA-ICP-MS) and metallomics. Metallomics 2010, 2 (2), 104-111. 68. Zvyagin, A. V.; Zhao, X.; Gierden, A.; Sanchez, W.; Ross, J. A.; Roberts, M. S., Imaging of zinc oxide nanoparticle penetration in human skin in vitro and in vivo. Journal of Biomedical Optics 2008, 13 (6). 69. Kusakabe, T.; Nakajima, K.; Suzuki, K.; Nakazato, K.; Takada, H.; Satoh, T.; Oikawa, M.; Kobayashi, K.; Koyama, H.; Arakawa, K.; Nagamine, T., The changes of heavy metal and metallothionein distribution in testis induced by cadmium exposure. Biometals 2008, 21 (1), 71-81. 70. Stoeckli, M.; Chaurand, P.; Hallahan, D. E.; Caprioli, R. M., Imaging mass spectrometry: A new technology for the analysis of protein expression in mammalian tissues. Nature Medicine 2001, 7 (4), 493-496. 71. Becker, J. S., Bioimaging of metals in brain tissue from micrometre to nanometre scale by laser ablation inductively coupled plasma mass spectrometry: State of the art and perspectives. International Journal of Mass Spectrometry 2010, 289 (2-3), 65-75. 72. Durrant, S. F., Laser ablation inductively coupled plasma mass spectrometry: achievements, problems, prospects. Journal of Analytical Atomic Spectrometry 1999, 14 (9), 1385-1403. 73. Austin, C.; Fryer, F.; Lear, J.; Bishop, D.; Hare, D.; Rawling, T.; Kirkup, L.; McDonagh, A.; Doble, P., Factors affecting internal standard selection for quantitative

165 Literature elemental bio-imaging of soft tissues by LA-ICP-MS. Journal of Analytical Atomic Spectrometry 2011, 26 (7), 1494-1501. 74. Zoriy, M. V.; Dehnhardt, M.; Matusch, A.; Becker, J. S., Comparative imaging of P, S, Fe, Cu, Zn and C in thin sections of rat brain tumor as well as control tissues by laser ablation inductively coupled plasma mass spectrometry. Spectrochimica Acta Part B 2008, 63 (3), 375-382. 75. Jackson, B.; Harper, S.; Smith, L.; Flinn, J., Elemental mapping and quantitative analysis of Cu, Zn, and Fe in rat brain sections by laser ablation ICP-MS. Analytical and Bioanalytical Chemistry 2006, 384 (4), 951-957. 76. Pugh, J. A. T.; Cox, A. G.; McLeod, C. W.; Bunch, J.; Whitby, B.; Gordon, B.; Kalber, T.; White, E., A novel calibration strategy for analysis and imaging of biological thin sections by laser ablation inductively coupled plasma mass spectrometry. Journal of Analytical Atomic Spectrometry 2011, 26 (8), 1667-1673. 77. Becker, J. S.; Zoriy, M. V.; Pickhardt, C.; Palomero-Gallagher, N.; Zilles, K., Imaging of copper, zinc, and other elements in thin section of human brain samples (Hippocampus) by laser ablation inductively coupled plasma mass spectrometry. Analytical Chemistry 2005, 77 (10), 3208-3216. 78. Austin, C.; Hare, D.; Rawling, T.; McDonagh, A. M.; Doble, P., Quantification method for elemental bio-imaging by LA-ICP-MS using metal spiked PMMA films. Journal of Analytical Atomic Spectrometry 2010, 25 (5), 722-725. 79. Hare, D.; Burger, F.; Austin, C.; Fryer, F.; Grimm, R.; Reedy, B.; Scolyer, R. A.; Thompson, J. F.; Doble, P., Elemental bio-imaging of melanoma in lymph node biopsies. Analyst 2009, 134 (3), 450-453. 80. Becker, J. S.; Breuer, U.; Hsieh, H. F.; Osterholt, T.; Kumtabtim, U.; Wu, B.; Matusch, A.; Caruso, J. A.; Qin, Z. Y., Bioimaging of Metals and Biomolecules in Mouse Heart by Laser Ablation Inductively Coupled Plasma Mass Spectrometry and Secondary Ion Mass Spectrometry. Analytical Chemistry 2010, 82 (22), 9528-9533. 81. Zoriy, M.; Matusch, A.; Spruss, T.; Becker, J. S., Laser ablation inductively coupled plasma mass spectrometry for imaging of copper, zinc, and platinum in thin sections of a kidney from a mouse treated with cis-platin. International Journal of Mass Spectrometry 2007, 260 (2-3), 102-106. 82. Wu, B.; Niehren, S.; Becker, J. S., Mass spectrometric imaging of elements in biological tissues by new BrainMet technique—laser microdissection inductively

166 BAM-Dissertationsreihe Literature coupled plasma mass spectrometry (LMD-ICP-MS). Journal of Analytical Atomic Spectrometry 2011, 26, 1653. 83. Hutchinson, R. W.; Cox, A. G.; McLeod, C. W.; Marshall, P. S.; Harper, A.; Dawson, E. L.; Howlett, D. R., Imaging and spatial distribution of beta-amyloid peptide and metal ions in Alzheimer's plaques by laser ablation-inductively coupled plasma-mass spectrometry. Analytical Biochemistry 2005, 346 (2), 225-233. 84. Seuma, J.; Bunch, J.; Cox, A.; McLeod, C.; Bell, J.; Murray, C., Combination of immunohistochemistry and laser ablation ICP mass spectrometry for imaging of cancer biomarkers. Proteomics 2008, 8 (18), 3775-3784. 85. Junqueira, L. C.; Carneiro, J.; Schiebler, T. H., Histologie. 4th ed.; Springer- Verlag Berlin Heidelberg: 1996. 86. Slamon, D. J.; Godolphin, W.; Jones, L. A.; Holt, J. A.; Wong, S. G.; Keith, D. E.; Levin, W. J.; Stuart, S. G.; Udove, J.; Ullrich, A.; Press, M. F., Studies of the Her- 2/Neu Proto-Oncogene in Human-Breast and Ovarian-Cancer. Science 1989, 244 (4905), 707-712. 87. Chu, P. G.; Wu, E.; Weiss, L. M., Cytokeratin 7 and cytokeratin 20 expression in epithelial neoplasms: A survey of 435 cases. Modern Pathology 2000, 13 (9), 962- 972. 88. Yang, E.; Hu, X. F.; Xing, P. X., Advances of MUC1 as a target for breast cancer immunotherapy. Histology and Histopathology 2007, 22 (8), 905-922. 89. Sauter, G.; Simon, R.; Hillan, K., Tissue microarrays in drug discovery. Nature Reviews Drug Discovery 2003, 2 (12), 962-972. 90. Grube, D., Constants and variables in immunohistochemistry. Arch Histol Cytol 2004, 67 (2), 115-134. 91. Gschwendtner, A.; Mairinger, T., How Thick Is Your Section - the Influence of Section Thickness on DNA-Cytometry on Histological Sections. Analytical Cellular Pathology 1995, 9 (1), 29-37. 92. Lajtha, A., Handbook of Neurochemistry and Molecular Neurobiology. 3rd ed.; Springer Science and Business Media: 2007. 93. Travis, W. D.; Brambilla, E.; Noguchi, M.; Nicholson, A. G.; Geisinger, K. R.; Yatabe, Y.; Beer, D. G.; Powell, C. A.; Riely, G. J.; Van Schil, P. E.; Garg, K.; Austin, J. H. M.; Asamura, H.; Rusch, V. W.; Hirsch, F. R.; Scagliotti, G.; Mitsudomi, T.; Huber, R. M.; Ishikawa, Y.; Jett, J.; Sanchez-Cespedes, M.; Sculier, J. P.; Takahashi,

167 Literature

T.; Tsuboi, M.; Vansteenkiste, J.; Wistuba, I.; Yang, P. C.; Aberle, D.; Brambilla, C.; Flieder, D.; Franklin, W.; Gazdar, A.; Gould, M.; Hasleton, P.; Henderson, D.; Johnson, B.; Johnson, D.; Kerr, K.; Kuriyama, K.; Lee, J. S.; Miller, V. A.; Petersen, I.; Roggli, V.; Rosell, R.; Saijo, N.; Thunnissen, E.; Tsao, M.; Yankelewitz, D., International Association for the Study of Lung Cancer/American Thoracic Society/European Respiratory Society International Multidisciplinary Classification of Lung Adenocarcinoma. Journal of Thoracic Oncology 2011, 6 (2), 244-285. 94. Isacke, C. M.; Robertson, D.; Savage, K.; Reis-Filho, J. S., Multiple immunofluorescence labelling of formalin-fixed paraffin-embedded (FFPE) tissue. Bmc Cell Biology 2008, 9. 95. Galanski, M.; Arion, V. B.; Jakupec, M. A.; Keppler, B. K., Recent developments in the field of tumor-inhibiting metal complexes. Current Pharmaceutical Design 2003, 9 (25), 2078-2089. 96. Todd, R. C.; Lippard, S. J., Inhibition of transcription by platinum antitumor compounds. Metallomics 2009, 1 (4), 280-291. 97. Rosenberg, B.; Vancamp, L.; Krigas, T., Inhibition of Cell Division in Escherichia Coli by Electrolysis Products from a Platinum Electrode. Nature 1965, 205 (4972), 698-699. 98. Perez, J. M.; Cepeda, V.; Fuertes, M. A.; Castilla, J.; Alonso, C.; Quevedo, C., Biochemical Mechanisms of Cisplatin Cytotoxicity. Anti-Cancer Agents in Medicinal Chemistry 2007, 7 (1), 3-18. 99. Esteban-Fernández, D.; Moreno-Gordaliza, E.; Cañas, B.; Palacios, M. A.; Gómez-Gómez, M. M., Analytical methodologies for metallomics studies of antitumor Pt-containing drugs. Metallomics 2010, 2 (1), 19-38. 100. Esteban-Fernández, D.; Verdaguer, J. M.; Ramirez-Camacho, R.; Palacios, M. A.; Gómez-Gómez, M. M., Accumulation, fractionation, and analysis of platinum in toxicologically affected tissues after cisplatin, oxaliplatin, and carboplatin administration. Journal of Analytical Toxicology 2008, 32 (2), 140-146. 101. Dong, Z.; Pabla, N., Cisplatin nephrotoxicity: Mechanisms and renoprotective strategies. Kidney International 2008, 73 (9), 994-1007. 102. Dolan, M. E.; Rabik, C. A., Molecular mechanisms of resistance and toxicity associated with platinating agents. Cancer Treatment Reviews 2007, 33 (1), 9-23.

168 BAM-Dissertationsreihe Literature

103. Nugent, K.; Yao, X.; Panichpisal, K.; Kurtzman, N., Cisplatin nephrotoxicity: A review. American Journal of the Medical Sciences 2007, 334 (2), 115-124. 104. Taylor, H. E., Inductively Coupled Plasma-Mass Spectrometry. Academic Press: San Diego, California, U.S.A., 2001. 105. Butler, O. T.; Cairns, W.; Cook, J. M.; Davidson, C. M., Atomic spectrometry update. Environmental analysis. Journal of Analytical Atomic Spectrometry 2011, 26 (2), 250-286. 106. Barefoot, R. R., Determination of platinum group elements and gold in geological materials: a review of recent magnetic sector and laser ablation applications. Analytica Chimica Acta 2004, 509 (2), 119-125. 107. Lange, B.; Recknagel, S.; Czerwensky, M.; Matschat, R.; Michaelis, M.; Peplinski, B.; Panne, U., Analysis of pure copper - a comparison of analytical methods. Microchimica Acta 2008, 160 (1-2), 97-107. 108. Careri, M.; Elviri, L.; Mangia, A., Element-tagged immunoassay with inductively coupled plasma mass spectrometry for multianalyte detection. Analytical and Bioanalytical Chemistry 2009, 393 (1), 57-61. 109. Sar, D. G.; Montes-Bayón, M.; González, E. B.; Zapico, L. M. S.; Sanz-Medel, A., Reduction of Cisplatin-Induced Nephrotoxicity in Vivo by Selenomethionine: The Effect on Cisplatin-DNA Adducts. Chemical Research in Toxicology 2011, 24 (6), 896-904. 110. Houk, R. S.; Fassel, V. A.; Flesch, G. D.; Svec, H. J.; Gray, A. L.; Taylor, C. E., Inductively Coupled Argon Plasma as an Ion-Source for Mass-Spectrometric Determination of Trace-Elements. Analytical Chemistry 1980, 52 (14), 2283-2289. 111. Rita Cornelis, R.; Caruso, J.; Crews, H.; Heumann, K., Handbook of Elemental Speciation John Wiley & Sons Ltd.: Chichester, West Sussex, England, 2003; Vol. 1. 112. Sharp, B. L., Pneumatic Nebulizers and Spray Chambers for Inductively Coupled Plasma Spectrometry - a Review .1. Nebulizers. Journal of Analytical Atomic Spectrometry 1988, 3 (5), 613-652. 113. Sharp, B. L., Pneumatic Nebulizers and Spray Chambers for Inductively Coupled Plasma Spectrometry - a Review .2. Spray Chambers. Journal of Analytical Atomic Spectrometry 1988, 3 (7), 939-963. 114. Agilent Technologies. http://www.chem.agilent.com/Library/primers/Public/ICP- MS_Primer-Web.pdf.

169 Literature

115. Gray, A. L., Solid Sample Introduction by Laser Ablation for Inductively Coupled Plasma Source-Mass Spectrometry. Analyst 1985, 110 (5), 551-556. 116. Traub, H.; Wälle, M.; Koch, J.; Panne, U.; Matschat, R.; Kipphardt, H.; Günther, D., Evaluation of different calibration strategies for the analysis of pure copper and zinc samples using femtosecond laser ablation ICP-MS. Analytical and Bioanalytical Chemistry 2009, 395 (5), 1471-1480. 117. Woodhead, J. D.; Hellstrom, J.; Hergt, J. M.; Greig, A.; Maas, R., Isotopic and elemental imaging of geological materials by laser ablation inductively coupled plasma-mass spectrometry. Geostandards and Geoanalytical Research 2007, 31 (4), 331-343. 118. Eggins, S. M.; Kinsley, L. P. J.; Shelley, J. M. G., Deposition and element fractionation processes during atmospheric pressure laser sampling for analysis by ICP-MS. Applied Surface Science 1998, 127, 278-286. 119. Houk, R. S.; Perdian, D. C.; Bajic, S. J.; Baldwin, D. P., Time-resolved studies of particle effects in laser ablation inductively coupled plasma-mass spectrometry. Journal of Analytical Atomic Spectrometry 2008, 23 (3), 336-341. 120. Koch, J.; Wälle, M.; Flamigni, L.; Heiroth, S.; Lippert, T.; Hartung, W.; Günther, D., Detection efficiencies in nano- and femtosecond laser ablation inductively coupled plasma mass spectrometry. Spectrochimica Acta Part B-Atomic Spectroscopy 2009, 64 (1), 109-112. 121. Kuhn, H. R.; Günther, D., Laser ablation-ICP-MS: particle size dependent elemental composition studies on filter-collected and online measured aerosols from glass. Journal of Analytical Atomic Spectrometry 2004, 19 (9), 1158-1164. 122. Hattendorf, B.; Latkoczy, C.; Günther, D., Laser ablation-ICPMS. Analytical Chemistry 2003, 75 (15), 341a-347a. 123. Garcia, C. C.; Lindner, H.; Niemax, K., Laser ablation inductively coupled plasma mass spectrometry-current shortcomings, practical suggestions for improving performance, and experiments to guide future development. Journal of Analytical Atomic Spectrometry 2009, 24 (1), 14-26. 124. Kuhn, H. R.; Günther, D., Elemental fractionation studies in laser ablation inductively coupled plasma mass spectrometry on laser-induced brass aerosols. Analytical Chemistry 2003, 75 (4), 747-753.

170 BAM-Dissertationsreihe Literature

125. Guillong, M.; Kuhn, H. R.; Günther, D., Application of a particle separation device to reduce inductively coupled plasma-enhanced elemental fractionation in laser ablation-inductively coupled plasma-mass spectrometry. Spectrochimica Acta Part B-Atomic Spectroscopy 2003, 58 (2-3), 211-220. 126. Guillong, M.; Günther, D., Effect of particle size distribution on ICP-induced elemental fractionation in laser ablation-inductively coupled plasma-mass spectrometry. Journal of Analytical Atomic Spectrometry 2002, 17 (8), 831-837. 127. Fernández, B.; Claverie, F.; Pécheyran, C.; Donard, O. F. X., Direct analysis of solid samples by fs-LA-ICP-MS. Trends in Analytical Chemistry 2007, 26 (10), 951- 966. 128. Koch, J.; Wälle, M.; Schlamp, S.; Rosgen, T.; Günther, D., Expansion phenomena of aerosols generated by laser ablation under helium and argon atmosphere. Spectrochimica Acta Part B-Atomic Spectroscopy 2008, 63 (1), 37-41. 129. Schena, M.; Shalon, D.; Davis, R. W.; Brown, P. O., Quantitative Monitoring of Gene-Expression Patterns with a Complementary-DNA Microarray. Science 1995, 270 (5235), 467-470. 130. Hegde, P.; Qi, R.; Abernathy, K.; Gay, C.; Dharap, S.; Gaspard, J. R.; Hughes, E.; Snesrud, E.; Lee, N.; Quackenbush, J., A Concise Guide to cDNA Microarray Analysis. BioTechniques 2000, 29, 548-562. 131. Shalon, D.; Smith, S. J.; Brown, P. O., A DNA microarray system for analyzing complex DNA samples using two-color fluorescent probe hybridization. Genome Research 1996, 6 (7), 639-645. 132. Chattopadhyay, P. K.; Price, D. A.; Harper, T. F.; Betts, M. R.; Yu, J.; Gostick, E.; Perfetto, S. P.; Goepfert, P.; Koup, R. A.; De Rosa, S. C.; Bruchez, M. P.; Roederer, M., Quantum dot semiconductor nanocrystals for immunophenotyping by polychromatic flow cytometry. Nature Medicine 2006, 12 (8), 972-977. 133. Wood, W. G., Immunoassays & Co.: Past, Present, Future? - A Review and Outlook from Personal Experience and Involvement over the Past 35 Years. Clinical Laboratory 2008, 54 (11-12), 423-438. 134. Ambrosius, H.; Luppa, H., Immunhistochemie, Grundlagen und Techniken. Akademie-Verlag Berlin: 1987. 135. Ward, P. A., Monoclonal Antibody Production. The National Academies Press: Washington D.C., 1999.

171 Literature

136. Singh, K. V.; Kaur, J.; Varshney, G. C.; Raje, M.; Suri, C. R., Synthesis and characterization of hapten-protein conjugates for antibody production against small molecules. Bioconjugate Chemistry 2004, 15 (1), 168-173. 137. Coons, A. H.; Creech, H. J.; Jones, R. N., Immunological properties of an antibody containing a fluorescent group. Proceedings of the Society for Experimental Biology and Medicine 1941, 47 (2), 200-202. 138. Vavilov, S. J. J., Collected Papers (in Russian). 1936, 1. 139. Hemmila, I.; Soini, E.; Lovgren, T., Time-Resolved Fluoroimmunoassay (Tr- Fia). Fresenius Zeitschrift Fur Analytische Chemie 1982, 311 (4), 357-357. 140. Yalow, R. S.; Berson, S. A., Immunoassay of Endogenous Plasma Insulin in Man. Journal of Clinical Investigation 1960, 39 (7), 1157-1175. 141. Brady, J. F., Mathematical Aspects of Immunoassays. In Immunoassay and other Bioanalytical Techniques, Van Emon, J. M., Ed. CRC Press: Boca Raton, FL, U.S.A., 2007. 142. Zeck, A.; Eikenberg, A.; Weller, M. G.; Niessner, R., Highly sensitive immunoassay based on a monoclonal antibody specific for [4-arginine]microcystins. Analytica Chimica Acta 2001, 441 (1), 1-13. 143. Ekins, R. P., The 'precision profile': its use in RIA assessment and design. Ligand Q. 1981, 4, 33-44. 144. Coons, A. H.; Creech, H. J.; Jones, R. N.; Berliner, E., The demonstration of pneumococcal antigen in tissues by the use of fluorescent antibody. Journal of Immunology 1942, 45 (3), 159-170. 145. Graham, R. C.; Karnovsk.Mj, Early Stages of Absorption of Injected Horseradish Peroxidase in Proximal Tubules of Mouse Kidney - Ultrastructural Cytochemistry by a New Technique. Journal of Histochemistry & Cytochemistry 1966, 14 (4), 291-302. 146. Tulchin, N.; Ornstein, L.; Davis, B. J., Microgel System for Disk- Electrophoresis. Analytical Biochemistry 1976, 72 (1-2), 485-490. 147. Cammann, K., Instrumentelle Analytische Chemie: Verfahren, Anwendungen und Qualitätssicherung. Spektrum Verlag: 2000. 148. Fazekas, S. D. S.; Webster, R. G.; Datyner, A., 2 New Staining Procedures for Quantitative Estimation of Proteins on Electrophoretic Strips. Biochimica Et Biophysica Acta 1963, 71 (2), 377-391.

172 BAM-Dissertationsreihe Literature

149. Switzer, R. C.; Merril, C. R.; Shifrin, S., Highly Sensitive Silver Stain for Detecting Proteins and Peptides in Polyacrylamide Gels. Analytical Biochemistry 1979, 98 (1), 231-237. 150. Diezel, W.; Kopperschläger, G.; Hofmann, E., Improved Procedure for Protein Staining in Polyacrylamide Gels with a New Type of Coomassie Brilliant Blue. Analytical Biochemistry 1972, 48 (2), 617-&. 151. Kolosova, A. Y.; Shim, W. B.; Kim, Y. J.; Yang, Z. Y.; Park, S. J.; Eremin, S. A.; Lee, I. S.; Chung, D. H., Fluorescence polarization immunoassay based on a monoclonal antibody for the detection of ochratoxin A. International Journal of Food Science and Technology 2004, 39 (8), 829-837. 152. Fricker, M. B.; Kutscher, D.; Aeschlimann, B.; Frommer, J.; Dietiker, R.; Bettmer, J.; Günther, D., High spatial resolution trace element analysis by LA-ICP- MS using a novel ablation cell for multiple or large samples. International Journal of Mass Spectrometry 2011. 153. GeoREM. http://georem.mpch-mainz.gwdg.de/. 154. Pérez, M.; Castilla, M.; Torres, A. M.; Lázaro, J. A.; Sarmiento, E.; Tejedor, A., Inhibition of brush border dipeptidase with cilastatin reduces toxic accumulation of cyclosporin A in kidney proximal tubule epithelial cells. Nephrology Dialysis Transplantation 2004, 19 (10), 2445-2455. 155. Klose, J.; Kobalz, U., 2-Dimensional Electrophoresis of Proteins - an Updated Protocol and Implications for a Functional-Analysis of the Genome. Electrophoresis 1995, 16 (6), 1034-1059. 156. Organization, W. H., Environmental health criteria 105: Selected mycotoxins: Ochratoxins, trichothecenes, ergot. Geneva: WHO. 1990. 157. Clark, H. A.; Snedeker, S. M., Ochratoxin A: Its cancer risk and potential for exposure. Journal of Toxicology and Environmental Health-Part B-Critical Reviews 2006, 9 (2-3), 265-296. 158. Mantle, P. G.; Nagy, J. M., Binding of ochratoxin a to a urinary globulin: A new concept to account for gender difference in rat nephrocarcinogenic responses. International Journal of Molecular Sciences 2008, 9 (5), 719-735. 159. Biro, K.; Solti, L.; Barna-Vetro, I.; Bago, G.; Glavits, R.; Szabo, E.; Fink- Gremmels, J., Tissue distribution of ochratoxin A as determined by HPLC and ELISA and histopathological effects in chickens. Avian Pathology 2002, 31 (2), 141-148.

173 Literature

160. Huff, J. E., Carcinogenicity of ochratoxin A in experimental animals. IARC Scientific Publications 1991, 115, 229-244. 161. Directive 1881/2006, Setting maximum levels for certain contaminants in foodstuffs. Official Journal of the European Union 19 December 2006. 162. Directive 105/2010, Amending Regulation (EC) No 1881/2006 Setting maximum levels for certain contaminants in foodstuffs as regards ochratoxin A. Official Journal of the European Union 5 February 2010. 163. González-Peñas, E.; Leache, C.; Viscarret, M.; de Obanos, A. P.; Araguás, C.; de Cerain, A. L., Determination of ochratoxin A in wine using liquid-phase microextraction combined with liquid chromatography with fluorescence detection. Journal of Chromatography A 2004, 1025 (2), 163-168. 164. Lindenmeier, M.; Schieberle, P.; Rychlik, M., Quantification of ochratoxin A in foods by a stable isotope dilution assay using high-performance liquid chromatography-tandem mass spectrometry. Journal of Chromatography A 2004, 1023 (1), 57-66. 165. Monaci, L.; Palmisano, F., Determination of ochratoxin A in foods: state-of-the- art and analytical challenges. Analytical and Bioanalytical Chemistry 2004, 378, 96– 103. 166. Beltrán, E.; Ibáñez, M.; Sancho, J. V.; Cortés, M. A.; Yusà, V.; Hernández, F., UHPLC-MS/MS highly sensitive determination of aflatoxins, the aflatoxin metabolite M1 and ochratoxin A in baby food and milk. Food Chemistry 2011, 126 (2), 737-744. 167. Lau, B. P. Y.; Scott, P. M.; Lewis, D. A.; Kanhere, S. R., Quantitative determination of ochratoxin A by liquid chromatography/electrospray tandem mass spectrometry. Journal of Mass Spectrometry 2000, 35 (1), 23-32. 168. Wendler, I.; Jentsch, K. D.; Schneider, J.; Hunsmann, G., Efficient Replication of Htlv-Iii and Stlv-Iiimac in Human Jurkat Cells. Medical Microbiology and Immunology 1987, 176 (5), 273-280. 169. Quinn, C. P.; Semenova, V. A.; Elie, C. M.; Romero-Steiner, S.; Greene, C.; Li, H.; Stamey, K.; Steward-Clark, E.; Schmidt, D. S.; Mothershed, E.; Pruckler, J.; Schwartz, S.; Benson, R. F.; Helsel, L. O.; Holder, P. F.; Johnson, S. E.; Kellum, M.; Messmer, T.; Thacker, W. L.; Besser, L.; Plikaytis, B. D.; Taylor, T. H.; Freeman, A. E.; Wallace, K. J.; Dull, P.; Sejvar, J.; Bruce, E.; Moreno, R.; Schuchat, A.; Lingappa, J. R.; Marano, N.; Martin, S. K.; Walls, J.; Bronsdon, M.; Carlone, G. M.; Bajani-Ari,

174 BAM-Dissertationsreihe Literature

M.; Ashford, D. A.; Stephens, D. S.; Perkins, B. A., Specific, sensitive, and quantitative enzyme-linked Immunosorbent assay for human immunoglobulin G antibodies to anthrax toxin protective antigen. Emerging Infectious Diseases 2002, 8 (10), 1103-1110. 170. Carvalho, J. J.; Weller, M. G.; U. Panne, U.; Schneider, R. J., A highly sensitive caffeine immunoassay based on a monoclonal antibody. Analytical and Bioanalytical Chemistry 2010, 396, 2617–2628. 171. Kull, S. Entwicklung eines Immunoassays für das Mykotoxin Ochratoxin A. Humboldt Universität zu Berlin, Berlin, 2007. 172. Porstmann, T.; Kiessig, S. T., Enzyme-Immunoassay Techniques - an Overview. Journal of Immunological Methods 1992, 150 (1-2), 5-21. 173. Giesen, C.; Jakubowski, N.; Panne, U.; Weller, M. G., Comparison of ICP-MS and photometric detection of an immunoassay for the determination of ochratoxin A in wine. Journal of Analytical Atomic Spectrometry 2010, 25 (10), 1567-1572. 174. Ribéreau-Gayon, P.; Glories, Y.; Maujean, A.; Dubourdieu, D., Handbook of Enology. 2006; Vol. 2. 175. Krüger, R., Bremser, W., Koch, M., Rasenko, T., Nehls, I. The certification of the mass content of ochratoxin A in red wine, certified reference material ERM®- BD476; 2010. 176. Bally, M.; Halter, M.; Voros, J.; Grandin, H. M., Optical microarray biosensing techniques. Surface and Interface Analysis 2006, 38 (11), 1442-1458. 177. Deegan, R. D.; Bakajin, O.; Dupont, T. F.; Huber, G.; Nagel, S. R.; Witten, T. A., Capillary flow as the cause of ring stains from dried liquid drops. Nature 1997, 389 (6653), 827-829. 178. Cai, H.; Wang, Y. Q.; He, P. G.; Fang, Y. H., Electrochemical detection of DNA hybridization based on silver-enhanced gold nanoparticle label. Analytica Chimica Acta 2002, 469 (2), 165-172. 179. Bonanni, A.; Esplandiu, M. J.; del Valle, M., Impedimetric genosensors employing COOH-modified carbon nanotube screen-printed electrodes. Biosensors & Bioelectronics 2009, 24 (9), 2885-2891. 180. Bleiner, D.; Günther, D., Theoretical description and experimental observation of aerosol transport processes in laser ablation inductively coupled plasma mass spectrometry. Journal of Analytical Atomic Spectrometry 2001, 16 (5), 449-456.

175 Literature

181. Pettke, T.; Heinrich, C. A.; Ciocan, A. C.; Günther, D., Quadrupole mass spectrometry and optical emission spectroscopy: detection capabilities and representative sampling of short transient signals from laser-ablation. Journal of Analytical Atomic Spectrometry 2000, 15 (9), 1149-1155. 182. Leach, A. M.; Hieftje, G. M., Standardless semiquantitative analysis of metals using single-shot laser ablation inductively coupled plasma time-of-flight mass spectrometry. Analytical Chemistry 2001, 73 (13), 2959-2967. 183. Felton, J. A.; Schilling, G. D.; Ray, S. J.; Sperline, R. P.; Denton, M. B.; Barinaga, C. J.; Koppenaal, D. W.; Hieftje, G. M., Evaluation of a fourth-generation focal plane camera for use in plasma-source mass spectrometry. Journal of Analytical Atomic Spectrometry 2011, 26 (2), 300-304. 184. Schilling, G. D.; Ray, S. J.; Rubinshtein, A. A.; Felton, J. A.; Sperline, R. P.; Denton, M. B.; Barinaga, C. J.; Koppenaal, D. W.; Hieftje, G. M., Evaluation of a 512- Channel Faraday-Strip Array Detector Coupled to an Inductively Coupled Plasma Mattauch-Herzog Mass Spectrograph. Analytical Chemistry 2009, 81 (13), 5467- 5473. 185. Giesen, C.; Mairinger, T.; Khoury, L.; Waentig, L.; Jakubowski, N.; and Panne, U., Multiplexed immunohistochemical detection of tumor markers in breast cancer tissue using laser ablation inductively coupled plasma mass spectrometry. Analytical Chemistry 2011, 83 (21), 8177-8183. 186. Brewer, G. J., Anticopper therapy against cancer and diseases of inflammation and fibrosis. Drug Discovery Today 2005, 10 (16), 1103-1109. 187. Zoriy, M. V.; Dehnhardt, M.; Reifenberger, G.; Zilles, K.; Becker, J. S., Imaging of Cu, Zn, Pb and U in human brain tumor resections by laser ablation inductively coupled plasma mass spectrometry. International Journal of Mass Spectrometry 2006, 257 (1-3), 27-33. 188. Todolí, J. L.; Mermet, J. M., Study of polymer ablation products obtained by ultraviolet laser ablation inductively coupled plasma atomic emission spectrometry. Spectrochimica Acta Part B-Atomic Spectroscopy 1998, 53 (12), 1645-1656. 189. Giesen, C.; Waentig, L.; Mairinger, T.; Drescher, D.; Kneipp, J.; Roos, P. H.; Panne, U.; Jakubowski, N., Iodine as an elemental marker for imaging of single cells and tissue sections by laser ablation inductively coupled plasma mass spectrometry. Journal of Analytical Atomic Spectrometry 2011, 26 (11), 2160–2165.

176 BAM-Dissertationsreihe Literature

190. Duncan, A. W.; Taylor, M. H.; Hickey, R. D.; Newell, A. E. H.; Lenzi, M. L.; Olson, S. B.; Finegold, M. J.; Grompe, M., The ploidy conveyor of mature hepatocytes as a source of genetic variation. Nature 2010, 467 (7316), 707-U93. 191. Meyer, K. A.; Ovchinnikova, O.; Ng, K.; Goeringer, D. E., Development of a scanning surface probe for nanoscale tip-enhanced desorption/ablation. Review of Scientific Instruments 2008, 79 (12), 123710,1-4. 192. Gammelgaard, B.; Jensen, B. P., Application of inductively coupled plasma mass spectrometry in drug metabolism studies. Journal of Analytical Atomic Spectrometry 2007, 22 (3), 235-249. 193. Raab, A.; Ploselli, B.; Munro, C.; Thomas-Oates, J.; Feldmann, J., Evaluation of gel electrophoresis conditions for the separation of metal-tagged proteins with subsequent laser ablation ICP-MS detection. Electrophoresis 2009, 30 (2), 303-314. 194. Camano, S.; Lazaro, A.; Moreno-Gordaliza, E.; Torres, A. M.; de Lucas, C.; Humanes, B.; Lazaro, J. A.; Gómez-Gómez, M. M.; Bosca, L.; Tejedor, A., Cilastatin Attenuates Cisplatin-Induced Proximal Tubular Cell Damage. Journal of Pharmacology and Experimental Therapeutics 2010, 334 (2), 419-429. 195. Gomez-Gomez, M. M.; Esteban-Fernandez, D.; Moreno-Gordaliza, E.; Canas, B.; Palacios, M. A., Analytical methodologies for metallomics studies of antitumor Pt- containing drugs. Metallomics 2010, 2 (1), 19-38. 196. Moreno-Gordaliza, E.; Giesen, C.; Lázaro, A.; Esteban-Fernández, D.; Humanes, B.; Cañas, B.; Panne, U.; Tejedor, A.; Jakubowski, N.; Gómez-Gómez, M. M., Elemental Bioimaging in Kidney by LA-ICP-MS As a Tool to Study Nephrotoxicity and Renal Protective Strategies in Cisplatin Therapies. Analytical Chemistry 2011, 83 (20), 7933-7940. 197. Dong, Z.; Pabla, N.; Murphy, R. F.; Liu, K. B., The copper transporter Ctr1 contributes to cisplatin uptake by renal tubular cells during cisplatin nephrotoxicity. American Journal of Physiology-Renal Physiology 2009, 296 (3), F505-F511. 198. Sharma, R. P., Interactions of Cis-Platinum with Cellular Zinc and Copper in Rat-Liver and Kidney Tissues. Pharmacological Research Communications 1985, 17 (2), 197-206. 199. Shannon, R. D., Revised Effective Ionic-Radii and Systematic Studies of Interatomic Distances in Halides and Chalcogenides. Acta Crystallographica Section A 1976, 32 (Sep1), 751-767.

177 Literature

200. Esteban-Fernández, D.; Cañas, B.; Pizarro, I.; Palacios, M. A.; Gómez- Gómez, M. M., SEC-ICP-MS and ESI-MS as tools to study the interaction between cisplatin and cytosolic biomolecules. Journal of Analytical Atomic Spectrometry 2007, 22 (9), 1113-1121. 201. Humanes, B.; Lazaro, A.; Camano, S.; Moreno-Gordaliza, E.; Lazaro, J. A.; Lara, J. M.; Ortiz, A.; Gómez-Gómez, M. M.; Martín-Vasallo, P.; Tejedor, A., Submitted to Kidney Int. 2011. 202. Tanner, M.; Günther, D., Short transient signals, a challenge for inductively coupled plasma mass spectrometry, a review. Analytica Chimica Acta 2009, 633 (1), 19-28. 203. Jeger, S.; Zimmermann, K.; Blanc, A.; Grunberg, J.; Honer, M.; Hunziker, P.; Struthers, H.; Schibli, R., Site-Specific and Stoichiometric Modification of Antibodies by Bacterial Transglutaminase. Angewandte Chemie-International Edition 2010, 49 (51), 9995-9997. 204. Garcia, C. C.; Murtazin, A.; Groh, S.; Becker, M.; Niemax, K., Characterization of particles made by desolvation of monodisperse microdroplets of analyte solutions and particle suspensions for nanoparticle calibration in inductively coupled plasma spectrometry. Spectrochimica Acta Part B-Atomic Spectroscopy 2010, 65 (1), 80-85. 205. Cima, I.; Schiess, R.; Wild, P.; Kaelin, M.; Schüffler, P.; Lange, V.; Picotti, P.; Ossola, R.; Templeton, A.; Schubert, O.; Fuchs, T.; Leippold, T.; Wyler, S.; Zehetner, J.; Jochum, W.; Buhmann, J.; Cerny, T.; Moch, H.; Gillessen, S.; Aebersold, R.; Krek, W., Cancer genetics-guided discovery of serum biomarker signatures for diagnosis and prognosis of prostate cancer. Proceedings of the National Academy of Sciences of the United States of America 2011, 108 (8), 3342-3347. 206. Yamashita, M.; Fenn, J. B., Electrospray Ion-Source - Another Variation on the Free-Jet Theme. Journal of Physical Chemistry 1984, 88 (20), 4451-4459. 207. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M., Electrospray Ionization-Principles and Practice. Mass Spectrometry Reviews 1990, 9 (1), 37-70. 208. Wilm, M. S.; Mann, M., Electrospray and Taylor-Cone Theory, Doles Beam of Macromolecules at Last. International Journal of Mass Spectrometry 1994, 136 (2-3), 167-180.

178 BAM-Dissertationsreihe Literature

209. Emmett, M. R.; Caprioli, R. M., Micro-Electrospray Mass-Spectrometry - Ultra- High-Sensitivity Analysis of Peptides and Proteins. Journal of the American Society for Mass Spectrometry 1994, 5 (7), 605-613. 210. Heck, A. J. R.; van den Heuvel, R. H. H.; van Duijn, E.; Mazon, H.; Synowsky, S. A.; Lorenzen, K.; Versluis, C.; Brouns, S. J. J.; Langridge, D.; van der Oost, J.; Hoyes, J., Improving the performance of a quadrupole time-of-flight instrument for macromolecular mass spectrometry. Analytical Chemistry 2006, 78 (21), 7473-7483. 211. Kneipp, J.; Kneipp, H.; McLaughlin, M.; Brown, D.; Kneipp, K., In vivo molecular probing of cellular compartments with gold nanoparticles and nanoaggregates. Nano Letters 2006, 6 (10), 2225-2231. 212. Uribe, M.; Fenogliopreiser, C. M.; Grimes, M.; Feind, C., Medullary Carcinoma of the Thyroid-Gland - Clinical, Pathological, and Immunohistochemical Features with Review of the Literature. American Journal of Surgical Pathology 1985, 9 (8), 577- 594.

179