MALDI Imaging Mass Spectrometry – a Novel Approach in Biomedical ...

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MALDI Imaging Mass Spectrometry – a Novel Approach in Biomedical Research of Tissues Magdalena Kalinowska-Herok1,2,*, Monika Pietrowska1, Anna Walaszczyk1 and Piotr Widak1 1

Center for Translational Research and Molecular Biology of Cancer Maria Sklodowska -Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland; 2PolishJapanese Institute of Information Technology Bytom Branch, Aleja Legionów 2, 41-902 Bytom, Poland Abstract: Matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) is a powerful tool for investigating the spatial distribution of proteins and small molecules within biological specimen. This technique combines the potential of MALDI-ToF mass spectrometry with the ability to scan series of pixels across the surface of tissues, which generates multiplex space-correlated mass spectra. Numerical processing of the data allows visualization of specific molecular species and their correlation with histological image of the tissue. Hence, IMS is a multiplex untargeted analysis that enables characterization of tissue regions based on their endogenous biomolecular content. The major advantage of this technique is its potential to define tissue regions based on their molecular profiles independently of their histological and morphological characteristics given by traditional tools. IMS can detect potential cancer foci within histologically normal tissue, reveal intra-tumor heterogeneity or altered protein expression at tumor/normal tissue interface zones. IMS is an emerging technology that could fill gaps in the knowledge of spatial nature of a disease allowing better understanding of connections between structure and molecular processes.

Keywords: MALDI-ToF mass spectrometry, molecular imaging, molecular histopathology, protein distribution, proteomics. INTRODUCTION TO SPECTROMETRY

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Mass spectrometry (MS) is a powerful and universal analytical technique capable to determine the mass of large biomolecular complexes, individual molecules or ions. Since its invention in the first decades of the 20th century mass spectrometry has undergone tremendous improvement in terms of mass ranges, resolution and sensitivity. As a result MS has an important place in many disciplines including chemistry, physics, biology, pharmacology and medicine. Among mass spectrometry techniques that had the major impact on protein science and proteomics was Matrix-Assisted Laser Desorption/Ionization (MALDI) MS coupled with Time-of-Flight (ToF) type of analyzer. In the MALDI-ToF technique proteins are mixed with solid matrix and then protonized after laser light-induced desorption from the matrix. Generated ions are accelerated in the electromagnetic field and the time of flight of individual ions before they hit the detector reflects their size (actually the mass to charge ratio) [1, 2], (Fig. 1A) presents scheme of MALDI-ToF analysis. Resulting spectra contain information about the size (m/z value) and abundance of analyzed ions. In the MALDI spectra measured m/z value of registered ions corresponds to molecular weight of ionized proteins/peptides (increased by *Addresses correspondence to this author at the Center for Translational Research and Molecular Biology of Cancer Maria Sklodowska -Curie Memorial Cancer Center and Institute of Oncology Gliwice Branch, Wybrzeze Armii Krajowej 15, 44-101 Gliwice, Poland; and Polish-Japanese Institute of Information Technology Bytom Branch, Aleja Legionów 2, 41-902 Bytom, Poland; Tel: +48 322789627; Fax: +48 322789840; E-mail: [email protected] 1570-1646/13 $58.00+.00

the mass of the proton; [M+H]+ ions). Described method in its basic version allows identification of characteristic features of the protein profiles of analyzed mixtures. In addition, MALDI-ToF spectrometry allows the identification of proteins based on the amino acid sequence in polypeptide chains. Most typically, analyzed protein is processed enzymaticaly (e.g., by trypsin digestion) then selected peptides further fragmented in spectrometer, and sizes (m/z values) of fragmentary ions are determined in the so called tandem mass spectrometry (MS/MS). On the basis of size of fragmentary ions the appropriate computational analysis allows the establishing of sequence of amino acids in the peptide chain and identification of corresponding protein [3, 4]. Most importantly, MALDI-ToF MS can be applied directly to samples of biological tissues. While traditional methods of protein analysis require homogenization of whole tissues, direct MS analysis of tissue requires much less sample manipulation and maintains the spatial integrity of the specimen. Thus, a single thin (~ 5-20
m) section of a tissue sample could be used not only for identification of proteins but also for determination of spatial distributions of detected molecules. Spatially-correlated MS analysis of tissue sample that can reveal abundance of different biomolecular ions in different sample spots is called imaging mass spectrometry, IMS. The schematic idea of imaging mass spectrometry is presented on (Figs. 1B and 1C). The idea of MALDI-IMS was first proposed in 1994 [5] and has been applied to visualize spatial distribution of peptides and proteins since 1997 [6]. Since that time MALDI-IMS has become a powerful technique that enables the identification and localization of different biological compounds including ©2013 Bentham Science Publishers

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Fig. (1). ????????????????????????????????????????.

proteins, lipids, pharmaceuticals and metabolites directly on tissue surfaces. The ability to determine the spatial distribution of biomolecules makes the MALDI-IMS a valuable tool in many fields of life sciences. In recent years IMS proved its potential in studies of human diseases, particularly in cancer research. Although different MS methods (including FTICR, ion-trap and Q-ToF) have been tried for imaging of small molecules [7-14]. MALDI-ToF mass spectrometry is the major method applied in imaging mass spectrometry, hence this review is focused on MALDI-IMS.

tially allow generating 3D molecular tissue images. The resulting images allow comparison of the distribution of molecules between different regions of the sample as well as between the samples [18]. The sample preparation methods are very similar for both types of imaging experiments, and the current matrix spotting instruments can print the matrix spots directly on a tissue section either as individual pre-selected spots for low resolution profiling or as dense spot arrays over the entire tissue for high resolution imaging [19, 20].

GENERAL STRATEGIES SPECTROMETRY

The experimental protocol of molecular imaging using MALDI-ToF mass spectrometry involves four general stages: (i) tissue sample preparation, (ii) matrix application, (iii) acquisition of spectra and (iv) data analysis. Procedures of tissue preparation and matrix application are the most crucial for successful implementation of MALDI-IMS specifically.

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There are two different approaches in the IMS type of experiments: profiling and classical tissue imaging. Usually, it is hard to obtain at the same time high spatial resolution, high speed as well as low cost of analysis, and unfortunatelly there is no golden standard. A compromise has to be made, sometimes through sacrifice of the spatial resolution. Low resolution imaging (profiling) involves sampling of discrete areas of the tissue sections. In a typical profiling experiment only few example spots (5-20 spots of approximately 1 mm in diameter) from the whole tissue section are analyzed. These experiments are designed to make comparisons between different types of tissue, e.g. normal versus cancer or untreated versus treated. In this type of analysis large numbers of samples could be analyzed, and then resulting mass profiles are subjected to statistical analyses to establish statistical significance of resulting classifications [15, 16]. Classical tissue imaging requires analysis of an entire tissue section through an ordered array of spots. In this case spectra are acquired at every 50-100
m in both directions of the section, and the resulting picture contains up to tens of thousands of pixels [17]. Molecular images, or 2D ion intensity maps, can be created by plotting the intensity of each registered ion as a function of its [x,y] coordinates. In addition, serial sections of tissues could be analyzed that would poten-

TISSUE PREPARATION FOR MALDI-IMS

The majority of reported IMS experiments has been performed using fresh snap-frozen tissue sections. In fact frozen chemically unmodified tissue represents the current gold standard for IMS. However, the limited availability and relatively short archival life of frozen tissue (maximum two years for proteomics applications) has forced researchers working with IMS to adopt methods of protein analysis from formalin-fixed paraffin embedded (FFPE) tissue. The FFPE standard is the current worldwide standard for long-term tissue preservation in medical and research centers [21-23], thus using this type of material would greatly enhance potential of IMS methods. However, formalin fixation induces cross-linking between multiple amino acid residues creating a linked protein network [24]. Due to protein cross-linking the IMS analysis of FFPE tissues is more complex and the protocols must include a cross-link reversal and paraffin removal steps. To access these tissues by MS, antigen retrieval (AR) [25, 26] and/or in situ tryptic digestion [27, 28] ir re-

MALDI Imaging Mass Spectrometry – a Novel Approach

quired. During this process, the mislocation of peptides is possible in the analyzed tissue. Therefore, it is always necessary to perform technical replicates as well as verify the IMS image in relation to histology. The AR step reverses crosslinks and denature linked proteins. However, this step could be insufficient for complete cross-link reversal and subsequent MS acquisition might not be of the same quality as generated using frozen tissue, hence AR is usually followed by tryptic digestion [30]. In addition the analysis of samples and data interpretation must consider the oxidation and degradation processes as well as effects of fixation and removal of paraffin on molecular structures [25, 28-31]. All these aspects must be taken into account especially when old samples (e.g., 100-year-old) are to be analyzed, and resulting data have to be dealt with a great caution [32]. More recently a new method of tissue fixation using RCL2®-CS100, which is a non-cross-linking fixative suitable for shotgun proteomic analyses and tissue imaging was proposed [33], which might overcome drawbacks of formalin fixation in future. Preparation of tissue sections requires the embedding of the tissue material in a supporting material, which allows handling and precise microtoming of sections. For routine histological applications tissues are usually embedded in the Optimal Cutting Temperature (O.C.T.) polymer. However, this type of polymer-based embedding media ionize easily during MS analysis and act as significant ion suppressors. To minimize this effect the use of ice or gelatin as embedding material is recommended [34]. Before acquisition of mass spectra tissue section has to be covered by a chemical matrix, which plays a key role in soft ionization characteristic for MALDI ion sources [1, 2]. Proteins are ionized in the co-crystals with the matrix molecules, which absorbs the laser energy and protects biomolecules from the disruptive energy. Mono-protonated molecular ions ([M+H]+) of peptides and proteins are generally detected in MALDI spectra. However, poli-protonated ions and sodium or potassium adduct ions ([M+Na/K]+) could be also detected in protein spectra. Hence selection of appropriate chemical formula of MALDI matrix is a very important issue for optimization of MALDI-IMS procedures. An overview of the matrices used for IMS can be found in many papers [35, 36]. The quality, robustness and reproducibility of MALDI-IMS frequently depends on protocols of the matrix application. Three types of matrix application methods are used. All of them requires constant temperature and low humidity to ensure homogeneous application of the matrix. The first method involves manual spraying using thin layer chromatography (TLC) sprayers or airbrushes [37]. This quick and inexpensive method allows coating of tissue sections with relatively small crystals, yet its apparent drawback is limited robustness and reproducibility. The second method based on automatic depositing of small droplets of matrix solution with robotic devices such as a chemical inkjet printer, which provide reproducible matrix preparations [38]. Compared to the droplet spot size in manual spraying, the droplet spot size in this method increases the signal sensitivity but decreases the spatial resolution (>100 μm) [39]. The third method is sublimation of the matrix under reduced pressure and elevated temperature [40-42]. This method requires no solvent; therefore, diffusion of the analyte mole-

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cules during matrix application is eliminated. Another advantage is the increased purity of the matrix and the formation of very small matrix crystals [43]. The IMS approach could be combined with different MSbased methods allowing identifying of registered protein, which significantly enhances potential of this technique. One of this research approaches is combination of MALDI-IMS with a laser capture microdissection (LCM), which is a cell isolation method based on microscopy-guided dissection of specific area of tissue section [44, 45]. Protein signatures identified by IMS could be used for selection of tissue regions for LCM-based cell isolation. The area for LCM might be delineated based on IMS annotation of adjacent tissue section, and then microdissected cells could be lyzed and proteins extracted, digested with trypsin and identified using LC-MS/MS strategies. A few reports described the combination of LCM and MS technologies for the analysis of different types of cells in breast cancer, including normal stroma and epithelial cells, and malignant invasive breast carcinoma cells. Such analysis revealed unique protein patterns from different cell subsets [28-31]. Another approach bases on the in-tissue digestion of proteins with trypsin. Resulting tryptic peptides are analyzed and identified by MALDI-ToF/ToF tandem MS directly from IMS plates [46]. Biological tissue represents an extremely challenging sample for direct MS analysis. The multiple molecules present in a tissue section (e.g., proteins, lipids, oligonucleotides, carbohydrates, small organic molecules and salts) can negatively influence each others desorption and ionization efficiency, hence disturbing their optimal detection. This phenomenon that could significantly limit the number of detected molecules is called ion(ization) suppression [4749]. Such ionization suppression occurs when one analyte is present in great excess or ionizes more easily than others. For example, MS spectra of highly vascularized tissues can be dominated by easily ionizing hemoglobin chains [42]. In addition lipids, carbohydrates and salts present in a tissue can promote adduct formation and affect cocrystallization of biomolecules with matrix, which also affects the quality of the mass spectra and number of registered proteins [46]. Albeit phenomenon of ionization suppression cannot be totally eliminated, optimizing of procedures of tissue preparation apparently limit its negative influence on quality and reliability of registered data. APPLICATION OF IMAGING MASS SPECTROMETRY IN CANCER RESEARCH MALDI-IMS was initially applied to tissue sections in 1997 [6] and since that time it has been tested in wide variety of biomedical problem. However, cancer has been so far the major focus of molecular imaging of tissues. In fact MALDIIMS has provided candidate cancer biomarkers that can be used to identify specific tissue regions, classify cancer types and differentiate normal/benign tissues from malignancy [50-52, 23]. IMS evidenced molecular intra-tumor heterogeneity indicating clonal development of cancer [53] and revealed malignancy-related proteins in histologically benign polyps [54]. In addition, IMS could be applied in monitoring of drug metabolism [50]. Classical method that allows assessing tissue distribution of proteins and other molecules is

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immunohistochemistry (IHC) reviewed elsewhere [55, 56]. The obvious advantage of IMS over IHC is that IMS is not a targeted approach limited to selected molecules recognized by specific antibodies. In addition IMS allows simultaneous analysis of the distribution of hundreds of biomolecules, which is not possible with IHC. IMS might be also particularly valuable when a lack of antibodies precludes protein identifications by IHC. Because IMS is a powerful tool for discovery of novel factors both IMS and IHC may have a complementary role in future molecular cancer histopathology. Currently the major focus of IMS has been protein profiling for cancer classification. Many different cancer types have been analyzed so far, including breast cancer [57, 24], prostate cancer [58-60], ovarian cancer [52], lung cancer [61], glioma [62, 63], head & neck cancer [64], and colon cancer metasteses in liver [65]. The objective of these comparative analyses was to distinguish cancer from normal tissue as well as classify different grades or subtypes of cancer. Potential of IMS-based cancer classification could be exemplified by a classic work of Yanagisawa and coworkers (2003), who analyzed molecular profiles of non-small cell lung cancer (NSCLC). Authors analyzed samples from 79 lung cancers and 14 normal lung tissues. Identified classifiers based on registered protein profiles allowed differentiation between primary NSCLC and normal lung or cancer metastases to lung, and to classify different histological subtypes of NSCLC (adenocarcinomas, squamous cell carcinomas and large cell carcinomas). Interestingly, that work evidenced correlation between prognosis and identified protein profiles. Classifier built of 15 registered protein peaks distinguished between patients with resected NSCLC who had poor prognosis (median survival 6 months) and those who had good prognosis (median survival 33 months) [61]. Another example for applicability of IMS-based protein profiling for prognosis of survival of cancer patient is metastatic melanoma. MALDI-IMS-based analysis of lymph nodes (metastatic and tumor-free) allowed identifying protein profiles that could be used for molecular sub-classification of patients and identification of signatures that correlated with either poor or good prognosis [66]. Discovery of a novel protein characteristic for ovarian cancer might be an interesting example of cross-validation of a marker candidate using different methods including IMS. In work of Lemaire and coworkers [52] advanced ovary carcinomas (25 samples) and benign ovaries (23 samples) were subjected to LC-MS/MS analyses aimed at identification of differentiating proteins. Analysis revealed differentiating peptide of 9744 Da that was identified as 84 amino acid residues peptide from the 11S proteasome activator complex, named PA28. Validation of this marker was performed using Western blot analysis and supplemented with MALDI-IMS and classical IHC. The analyses revealed epithelial expression of the protein with a nuclear localization in benign epithelium and a cytoplasmic localization in carcinomas [52]. An interesting cancer-related topic that could be successfully addressed by IMS is characterization of protein profiles at the interface zones between malignant and normal tissue. During last years (starting from the end of 90-ties) several papers have been published documenting that certain tumorspecific molecular factors spread beyond histologically de-

Kalinowska-Herok et al.

termined tumor borders into surrounding tissue. Such particular area of “morphologically normal” tissue is called molecular margin of cancer. Majority of published studies focused on specific proteins that were typically over-expressed (e.g., eIF4E) and/or mutated (e.g., p53) in cancer cells [6769], as well as on expression pattern, mutations and methylation status of cancer-specific genes [70-72]. Importantly, all these studies confirmed that positive molecular margins detected after tumor resection were predictive for loco-regional failure, while patients with negative molecular margins had much lower risk of cancer recurrence. Majority of these works took advantage of protein- and gene-specific tools (i.e., IHC and PCR), which approach was justified in diagnostics-oriented research. Molecule-targeted approaches are less favorable for general characterization of molecular events preceding morphological changes during malignant transformation. Only very recently high throughput methods of genomics/proteomics, including MALDI-IMS, were applied in a few studies of molecular cancer margins. First of such works addressed invasive soft tissue sarcoma [73]. The authors have shown that although marked differences in protein distribution between tumor and immediate adjacent tissue are observed several molecules characteristic for cancer persist far into histologically normal adjacent tissue. Several detected proteins showed abnormal levels of expression up to ~1.5 cm beyond the histological margin. A few of tumor-characteristic proteins were identified by LC-MS/MS and their spread into cancer margins was confirmed by IHC; among these proteins were calcyclin, calgranulin and macrophage migration inhibition factor. Similar analysis of renal carcinoma evidenced that among proteins that showed similar expression in tumor and its molecular margin were components of the mitochondrial electron transport system [74]. Another IMS-based analysis of ovarian cancer showed that in the interface zone between tumor and adjacent tissue the expression pattern of certain proteins could be distinct from both cancer and normal tissue; among proteins specific for such interface two were identified: plastin-2 and peroxiredoxin-1 [75]. Most interestingly, all this IMS-based analyses of cancer margins revealed ability to detect proteins that were not obvious cancer-specific molecules, hence showing potential to identify novel proteins involved in development of cancer and interactions between tumor and surrounding tissue. We can expect that future analyses of molecular margins of cancer will deliver more complete information about proteome features specific for this zone, hence enabling identification of molecular events and processes essential for development of cancer and its interactions with adjacent tissues. CONCLUDING REMARKS Imaging mass spectrometry represents a new analytical tool that provides direct information on spatial distribution and relative abundance of proteins in a tissue. The successful application of IMS for cancer classification, detection of intra-tumor heterogeneity and molecular tumor margins has been demonstrated for different types of malignancies. The capabilities of IMS offer enormous potential to identify disease-specific molecular events that occur prior to or without

MALDI Imaging Mass Spectrometry – a Novel Approach

morphological change, and to investigate changes for which other specific detection tools (e.g. immune-staining) are unavailable. Imaging mass spectrometry could be used to track molecular changes across tumors as well as their interfaces with normal tissue to determine the importance of specific protein or peptide species as cancer marker candidates. Following selection of such candidates identification of specific proteins can be achieved using different proteomics tool. After their validation (e.g. with IHC) such proteins could be used as diagnostic biomarkers in large patient cohorts studies. Additionally, the imaging mass spectrometry could fill gaps in our knowledge of cancer and allow better understanding connections between structure and molecular processes

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CONFLICT OF INTEREST The authors confirm that this article content has no conflicts of interest.

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ACKNOWLEDGEMENTS This work was supported by European Community from the European Social Fund within the INTERKADRA project UDA - POKL-04.01.01-00-014/10-00 (to MK-H) and the National Science Centre (grant no. 2011/03/D/NZ4/03507).

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