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MALDI Imaging Mass Spectrometry and Glycoprotein Biomarkers of Cancer - In my laboratory, a variety of mass spectrometry tools within the MUSC Proteomics Center and Mass Spectrometry Facility are being applied to cancer diagnosis and prognosis research. Our emphasis is the application of these tools for analysis of clinically derived tissues, fluids and exosomes to develop proteomic-based or small molecule diagnostic assays, with a particular focus on glycosylation and its role in disease. A new state-of-the-art, translational MALDI Imaging Research Center has been created. The facility is anchored by a dual-source Bruker Daltonic 7T Solarix FTICR mass spectrometer, two Bruker AutoFlex III MALDI instruments, ImagePrep sprayer and nanoLiter Cool Wave spotter. These are complemented by a Thermo Orbitrap Elite mass spectrometer and an Agilent Triversa Nanomate with LESA-MS for direct analysis of post-translational modifications to proteins. This instrumentation is being applied to multiple translational cancer research projects within my laboratory, as well as collaborations within the department and throughout the NCI-designated Hollings Cancer Center . These projects combine the capabilities of the MUSC Proteomics Center with clinical research, lipidomic, biorepository and molecular pathology resources. The current cancers being targeted are primarily prostate, kidney and pancreatic cancers. The major research areas of the Drake laboratory are summarized as follows.
MALDI Mass Spectrometry Tissue Imaging of Proteins, Glycans, Lipids and Drug Metabolites - MALDI imaging mass spectrometry (MALDI-IMS) is an established method used primarily to spatially profile proteins, lipids, drug and small molecule metabolites in tissues. Using the resources of the MUSC Proteomics Center and MALDI Imaging Research Center, our group has recently developed a glycobiology approach using on-tissue peptide N-glycanase F (PNGaseF) digestion to directly profile released N-linked glycans in their local microenvironment. Glycan profiling has been successfully used with fresh frozen and formalin-fixed tissues, and more recently, adapted to tissue microarrays representing prostate, lung, breast and pancreatic cancers. Using the dual-source Solarix FTICR MS instrumentation, another research emphasis is the development of methods to spatially profile bioactive ceramide, sphingosine and glycolipid specie distribution in tissues of clinical interest. A reference database of ceramide and glycosphingolipid masses for use in tissue imaging analysis has been created using tissues derived from lysosomal storage disease models (in collaboration with Jeffrey Medin, Univ. of Toronto and Yusuf Hannun, Stonybrook University), Drug uptake and metabolism in tissues derived from pre-clinical model systems is also ongoing, working in conjunction with researchers in the Hollings Cancer Center and the Developmental Cancer Therapeutics program. Using these approaches, a global snapshot of major cellular glycan, lipid and protein components is obtained, which includes their tissue localization and distribution, structure and relative concentration. The preparation and analysis workflows are readily linked to standard histopathology procedures. The method has been applied to multiple frozen tissues types, with emphasis on human renal, prostate and lung cancer tissues matched for tumor and non-tumor pieces, and tissues from different therapeutic mouse xenograft models of cancer. For clear cell renal cancer tissues, defining biomolecules that are unique to the non-tumor and tumor margin interface are being assessed. Current MALDI imaging analysis of these margin regions have identified multiple glycan, phospholipid, ceramide and protein species differentially expressed relative to non-tumor and tumor regions. Linking these biomolecular differences to known mediators of epithelial-to-mesenchymal transitions, pathology correlates and tumor metastasis are ongoing. The methods are also being applied to non-small cell lung carcinoma tissue, and a unique cohort of prostate tissues from an ongoing Vitamin D supplementation study prior to prostatectomy.
Identification of Prostate and Lung Cancer Glycoprotein Biomarkers - Our overall research goal is the identification of glycoprotein biomarkers that will improve clinical decision-making in the detection and management of prostate and lung cancers. My laboratory has been part of a systemic effort to characterize and identify potential biomarker targets in proximal fluids of the prostate, including direct prostatic fluids obtained just prior to prostatectomy, and expressed prostatic secretions in urine obtained in the urology clinic following digital rectal exam. Our recent publications cumulatively describe the identification of over 1100 proteins identified in post-DRE expressed prostatic secretions in urine (EPS-urine) and prostatic fluids termed direct EPS obtained at the time of prostatectomy. Lastly, we have completed the proteome analysis of exosomes from pooled EPS samples from benign, indolent and aggressive cancers. These cumulative lists are near complete and available for rapid glycoprotein candidate screening in future studies. For lung cancers, NSCLC tissue and bronchoalveilar lavage samples were obtained from the Lung Cancer Biospecimen Resource Network and Dr. Chad Denlinger at MUSC New HCD-ETD workflows for direct analysis of EPS and bronchoalveolar lavage fluids, exosomes and tissue glycopeptides associated with disease status are in progress. Corresponding glycomic and lipidomic analyses of the same fluids and samples are also ongoing.
A complementary biomarker discovery approach is being done using metabolic labeling with sugar-azides and alkyne-bead click chemistry to capture secreted and cell surface glycoproteins in human-derived prostate and lung cancer cell line models. A current emphasis is with cell lines associated with the epithelial-to-mesenchymal cell transition. The current workflow uses N-acetylgalactosamine (GalNAc-Az) or sialic acid (ManNAc-Az) analogs for metabolic labeling, cell lysis or concentration of conditioned media, and Click chemistry conjugation to alkyne beads. The bead-bound azide-modified glycoproteins can be extensively washed prior to direct protease digestion on the beads, which significantly reduces the background of non-specific, unmodified proteins identified at the mass spectrometry stage. Captured glycoproteins are digested directly from the beads and the resulting peptides analyzed on the Orbitrap Elite for protein identification and development of HCD-ETD glycopeptide analysis workflows. Bioinformatic pathway analysis is then done to link the proteins to molecular function and cancer biology. This biomarker selection step is being coupled with the EPS protein lists, and more recently bronchoalveolar lavage fluids, as part of a further biomarker candidate verification strategy.
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