Colon Tumor Biomarkers-Maldi Imaging of Tissue Microarray

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Purpose: Imaging MALDI (IMS) demonstrated the same colon tumor proteins, gi|119592539 hCG1787564[Homo sapiens] Mass: 57590 and gi|119592490 hCG2040674[Homo sapiens] Mass: 108178, in consecutive patients. The proteins were present in the tumors and normal satellite tissue. The presence of these proteins in the tumor and normal tissue raised several questions. Is this evidence of metastatic disease or spread of tumor into normal satellite tissue? Are these proteins biomarkers of field cancerization or a field defect, e.g., age-related hypermethylation in normal colonic mucosa? Does the use of histopathology alone, underestimate the extent of potential malignant disease? We hypothesized that histopathology, in combination with IMS, may identify metaplastic disease beyond the recognized tumor. To test this hypothesis we examined tissue microarrays of multiple colon tumors. Methods: Tissue microarrays were constructed from colon carcinomas blocks. The tumor microarray from each patient included three components: the tumor, surrounding satellite tissue, and normal control tissue. Contiguous histologic sections were obtained for IMS,3, histology,1, and protein extraction,1. The paraffin was removed and the paraformaldeyde induced protein cross-links reversed by heating the sections to 90° C for 15 minutes. The histologic sections were stained with hematoxylin and eosin. The respective MALDI matrices: sinapic acid, alpha cyano 4-hydroxy cinnamic acid, and 2, 5-dihydroxybenzoic acids were applied by sublimation to the 3 IMS sections. MALDI images and protein masses were obtained on a Shimadzu Axima TOF2 mass spectrometer. The third section was used for high pressure protein extraction with a Pressure BioSciences Barocycler. The extract was separated with nanoflow Liquid Chromatograph Mass Spectrometry (LCMS) and split into two aliquots. One aliquot was trypsinized, and processed for bottom-up protein identification with a nanoflow LCMS, Hitachi NanoFrontier. The second intact protein aliquot was processed directly for top-down protein identification with LCMS. Results: The high pressure extraction provided novel results. The protein yield was increased. New and larger numbers of proteins were extracted. The trypsin digest time was decreased from 12 hours to 45 minutes, and less trypsin was required for the digest. Tissue microarray IMS displayed the loci of proteins in tumor, tumor satellite tissue, and normal tissue, and allowed separation of the tumors into groups distinguished by the unique proteins from each group. The extraction experiments confirmed the proteins identified on IMS. Conclusion: Tissue MALDI can separate colon tumors by protein profiles and identify those tumors with concordant proteins in tumor satellite tissue.