Title: Mass Spectrometry-Based Intact Glycoproteomic Analysis of Human Clinical Samples for Novel Cancer Diagnostics
Abstract: Despite the well-known fact that glycosylation is aberrant in cancer, the link between glycan and protein information remains relatively poorly understood due to the analytical challenges associated with studying the complex modification in biological samples. In recent years, mass spectrometry has emerged as a preeminent tool to study protein glycosylation, and has enabled the characterization of intact glycopeptides, thereby allowing retention of information about both the glycan and modified protein. We have developed intact glycoproteomic analysis workflows and applied them to a variety of human clinical samples including tissue, urine, and other fluids, to identify, quantify, and characterize sites of protein glycosylation in a systematic fashion. Our approach includes protein extraction, enzymatic digestion, and glycopeptide enrichment using strong anion exchange with electrostatic repulsion hydrophilic interaction chromatography prior to analysis by LC-MS/MS on an Orbitrap mass spectrometer. In a recent glycopeptide analysis of clear cell renal cell carcinoma tissue samples, we identified more than 120,000 unique high confidence glycopeptides from more than 2000 glycoproteins. Protein glycosites were classified by glycan subtype as either complex (with or without sialic acids and/or fucose), high mannose, or hybrid. The diversity of glycosylation was compared between cancer and patient-matched adjacent non-cancer kidney tissue. Notably increased heterogeneity in glycosylation was observed in cancer compared to non-cancer tissue for specific glyosites and proteins, yielding intriguing information about the biological pathways altered by glycosylation in cancer. Overall results and specific examples will be presented. Results from applying our workflows to pancreatic cyst fluid and prostate cancer urine samples will also be discussed.
Bio: Dr. Sharon Pitteri is an Associate Professor of Radiology at Stanford University in the School of Medicine. She is also the Associate Chair of Research Operations for the Department of Radiology. Dr. Pitteri is a member of the Canary Center at Stanford for Cancer Early Detection, Stanford Cancer Institute, Stanford Bio-X Program, and Stanford Cancer Biology Program. She received her bachelor’s degree in Chemistry from Carleton College and her PhD in Chemistry from Purdue University advised by Dr. Scott McLuckey. Dr. Pitteri did her postdoctoral research in Molecular Diagnostics at the Fred Hutchinson Cancer Research Center. Her current research focuses on the identification of proteins and other molecules that are indicative of early stage and/or aggressive cancer in blood, tissue, and proximal fluids. Her lab develops and applies mass spectrometry-based methods to study protein glycosylation in order to better understand cancer biology and ultimately improve cancer diagnosis and therapeutics. She is the recipient of numerous research awards including the American Society for Mass Spectrometry Research Award, California Breast Cancer Research Program Innovative Development and Exploratory Award, Stanford McCormick Faculty Award, and Department of Defense Breast Cancer Breakthrough Award. She is also currently serves as Co-Chair of the steering committee of the National Cancer Institute (NCI) Alliance of Glycobiologists for Cancer Research and a standing member of the NIH Enabling Bioanalytical and Imaging Technologies Study Section. She was also elected as the Chair of the State of California Breast Cancer Research Program Breast Cancer Council and Treasurer of the American Society of Mass Spectrometry (ASMS) Board of Directors. Lastly, Dr. Pitteri is passionate about mentorship of students and postdoctoral scholars and serves as the Faculty Lead of the Stanford Propel Postdoc Scholars Program in the School of Medicine.
Host: Prof. Lingjun Li