Glycomic and Proteomic Profiling of Pancreatic Cyst Fluids Identifies Hyperfucosylated Lactosamines on the N-linked Glycans of Overexpressed Glycoproteins

Abstract

Pancreatic cancer is now the fourth leading cause of cancer deaths in the United States, and it is associated with an alarmingly low 5-year survival rate of 5%. However, a patient's prognosis is considerably improved when the malignant lesions are identified at an early stage of the disease and removed by surgical resection. Unfortunately, the absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas often prevents early detection of pancreatic cancer. To aid in the development of a molecular screening system for early detection of the disease, we have performed glycomic and glycoproteomic profiling experiments on 21 pancreatic cyst fluid samples, including fluids from mucinous cystic neoplasms and intraductal papillary mucinous neoplasms, two types of mucinous cysts that are considered high risk to undergo malignant transformation. A total of 80 asparagine-linked (N-linked) glycans, including high mannose and complex structures, were identified. Of special interest was a series of complex N-linked glycans containing two to six fucose residues, located predominantly as substituents on ␤-lactosamine extensions. Following the observation of these "hyperfucosylated" glycans, bottom-up proteomics experiments utilizing a label-free quantitative approach were applied to the investigation of two sets of tryptically digested proteins derived from the cyst fluids: 1) all soluble proteins in the raw samples and 2) a subproteome of the soluble cyst fluid proteins that were selectively enriched for fucosylation through the use of surface-immobilized Aleuria aurantia lectin. A comparative analysis of these two proteomic data sets identified glycoproteins that were significantly enriched by lectin affinity. Several candidate glycoproteins that appear hyperfucosylated were identified, including triacylglycerol lipase and pancreatic ␣-amylase, which were 20-and 22-fold more abundant, respectively, following A. aurantia lectin enrichment. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.015792, 1-11, 2012.Pancreatic cancer is associated with a poor long term outcome with an actual 5-year survival rate of less than 5%. The absence of a practical screening strategy and clinical diagnostic test for identifying premalignant lesions within the pancreas prevents early detection of pancreatic cancer. Detection of early stage pancreatic cancer will likely result from novel clinical methods aimed at early tumor detection and molecular profiling of individuals at high risk for pancreatic carcinogenesis.High-resolution imaging techniques have increased the detection of small, organ-based lesions in the intra-abdominal cavity. Incidental cysts within the pancreas are detected in ϳ13% of patients undergoing cross-sectional imaging for nonspecific abdominal symptoms (1). Greater than 80% of these incidental cysts are non-neoplastic, so few carry malignant potential. Pancreatic cysts are divided into three broad pathologic entities: serous cystadenomas (SCA), 1 mucinous ...

Discovery and Validation of Biomarkers That Distinguish Mucinous and Nonmucinous Pancreatic Cysts

“…Despite these efforts, MS-based approaches are being used more often to identify small molecules rather than protein markers. In fact, only a few proteomic investigations of pancreatic cyst fluids have been applied to overcome the current diagnostic limitations (11,13,15) for the differential diagnosis of cysts and early prediction of pancreatic cancer. Nevertheless, no reliable biomarkers have been discovered that can discriminate MC with malignant potential from NMC.…”

“…On the other hand, a proteomic analysis would require only small amounts of cyst fluid for testing, which would be a distinct advantage. Several proteins have been suggested as potential biomarkers to differentiate potentially malignant MC from NMC (11)(12)(13)(14)(15). Previous work on the global proteome of pancreatic cysts using mass spectrometry has discovered novel biomarkers and several biomarker candidates (e.g., olfactomedin-4, mucin-18, and solubilized CEA-related cell adhesion molecules) that were differentially expressed in malignant lesions.…”

New Trends in the Study of Protein Glycosylation in Oncological Diseases

“…tekutiny z cyst [37] nebo buněčné nádorové linie [38], je však potřeba modifi kovat protokol extrakce. povrchů a metod imobilizace, byly v literatuře již prodiskutovány [53][54][55].…”

Glycoprotein Disease Markers and Single Protein-omics

“…The variety of glycan structures on human proteins is not entirely known, although some estimates of the numbers exist (17). Each glycosylation site is typically occupied by a few major and additional minor glycan forms (18,19). In addition, the occupancy of each site, i.e.…”