Imaging mass spectrometry in papillary thyroid carcinoma for the identification and validation of biomarker proteins.

Direct tissue imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization and time-of-flight (MALDI-TOF) mass spectrometry has become increasingly important in biology and medicine, because this technology can detect the relative abundance and spatial distribution of interesting proteins in tissues. Five thyroid cancer samples, along with normal tissue, were sliced and transferred onto conductive glass slides. After laser scanning by MALDI-TOF equipped with a smart beam laser, images were created for individual masses and proteins were classified at 200-µm spatial resolution. Based on the spatial distribution, region-specific proteins on a tumor lesion could be identified by protein extraction from tumor tissue and analysis using liquid chromatography with tandem mass spectrometry (LC-MS/MS). Using all the spectral data at each spot, various intensities of a specific peak were detected in the tumor and normal regions of the thyroid. Differences in the molecular weights of expressed proteins between tumor and normal regions were analyzed using unsupervised and supervised clustering. To verify the presence of discovered proteins through IMS, we identified ribosomal protein P2, which is specific for cancer. We have demonstrated the feasibility of IMS as a useful tool for the analysis of tissue sections, and identified the tumor-specific protein ribosomal protein P2. Graphical Abstract Keywords: Pathology, Proteins, Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization, Thyroid Gland, Neoplasms INTRODUCTION Papillary thyroid carcinomas (PTC) are the most common form of thyroid cancer, accounting for nearly 85% of primary thyroid malignancies. Commonly, the solitary nodule is a palpably discrete swelling within an otherwise apparently normal thyroid gland, and is incidentally detected by radiological evaluation (1). From a clinical standpoint, the possibility of neoplastic disease is a major cause for concern in patients who present with thyroid nodules, because most conventional papillary carcinomas present as asymptomatic thyroid nodules. Recent studies have demonstrated 2 molecular mechanisms that function in the carcinogenesis of thyroid cancer: the mitogen-activated protein kinase (MAPK) and phosphatidylinositol-3-kinase (PI3K)/Akt pathways (2, 3). Additionally, such pathways are related to genetic alterations such as rearrangements in the "rearranged during transfection" (RET) or neurotrophic tyrosine kinase receptor 1 (NTRK1) genes, and the activation of point mutations in the BRAF gene (4). In a previous study, protein expression patterns according to genetic variables have been demonstrated to correlate with the microscopic features, clinical manifestations, and prognostic characteristics of PTC (5). Although various protein alterations have been detected as diagnostic markers or prognostic predictors, the challenge remains to identify the expression of new molecules in tumors. In biological research, protein expression profiling technology is a useful method to identify differential protein expression patterns and modifications. Analytical techniques with high sensitivity and increased throughput are required for the discovery of new biomarkers and new drugs. Recent progress in imaging mass spectrometry (IMS) has made it possible to identify several cellular components such as proteins, drugs, and other endogenous molecules directly on tissue sections (6, 7, 8, 9, 10). IMS uses matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) technology. The matrix is applied on cryosectioned tissue and a very small area of the matrix-applied tissue is analyzed using MALDI-MS. Differences between the analyzed areas are displayed by the imaging program. Each analyzed area of the tissue section, which can be as small as >200 µm, on a conductive surface such as gold-plated or indium tin oxide (ITO)-coated slide, is analyzed spot by spot using MALDI-MS after the application of the matrix. Using all the spectral data from each spot, the magnitudes of specific peaks on each spot can be displayed in terms of color intensity. In this way, spatial information on the tissue can be obtained. Based on the spatial distribution, region-specific proteins on a tissue can be identified by extracting proteins from the tissue, digesting them with trypsin, and analyzing the fragments using liquid chromatography with tandem MS (LC-MS/MS). This technology using IMS has broad applications in the detection of new proteins in various fields. Compared with conventional imaging methods, the advantages of IMS are that it does not require the additional use of a specific antibody against the protein, and that it integrates histopathology and protein expression (11). For this reason, IMS provides superior information regarding distinct molecular arrangements in tissue sections. In the present study, we attempted to analyze thyroid samples, including those from PTC, using IMS. Before analysis using IMS, the diagnosis was confirmed by microscopic evaluation, immunohistochemical (IHC) staining of markers such as CK19, galectin-3, and RET, and pyrosequencing of the BRAF mutation, and then 5 homogenous samples of PTC were selected. After comparison of the differential molecular weight distribution between normal and tumor tissues using IMS, a protein of a specific molecular weight was identified by sequencing combined with LC-MS/MS, and then the presence of this protein was reconfirmed by western blot analysis.