Glycoprotein Microarrays with Multi-Lectin Detection:  Unique Lectin Binding Patterns as a Tool for Classifying Normal, Chronic Pancreatitis and Pancreatic Cancer Sera

Abstract

Pancreatic cancer is the fourth leading cause of cancer-related death in the United States, with a 5-year survival rate of less than 4%. Effective early detection and screening are currently not available, and tumors are typically diagnosed at a late stage, frequently after metastasis. Existing clinical markers of pancreatic cancer lack specificity, as they are also found in inflammatory diseases of the pancreas and biliary tract. In the work described here, naturally occurring glycoproteins were enriched by using lectin affinity chromatography and then further resolved by nonporous reversed-phase chromatography. Glycoprotein microarrays were then printed and probed with a variety of lectins to screen glycosylation patterns in sera from normal, chronic pancreatitis, and pancreatic cancer patients. Ten normal, 8 chronic pancreatitis, and 6 pancreatic cancer sera were investigated. Data from the glycoprotein microarrays were analyzed using bioinformatics approaches including principal component analysis (PCA) and hierarchical clustering (HC). Both normal and chronic pancreatitis sera were found to cluster close together, although in two distinct groups, whereas pancreatic cancer sera were significantly different from the other two groups. Both sialylation and fucosylation increased as a function of cancer on several proteins including Hemopexin, Kininogen-1, Antithrombin-III, and Haptoglobin-related protein, whereas decreased sialylation was detected on plasma protease C1 inhibitor. Target alterations on glycosylations were verified by lectin blotting experiments and peptide mapping experiments using microLC-ESI-TOF. These altered glycan structures may have utility for the differential diagnosis of pancreatic cancer and chronic pancreatitis and identify critical differences between biological samples from patients with different clinical conditions.

The Prevalence and Nature of Glycan Alterations on Specific Proteins in Pancreatic Cancer Patients Revealed Using Antibody-Lectin Sandwich Arrays

“…Lectin binding patterns do not give precise information on glycan structures so it will be necessary in future studies to relate these data to results from other structural analysis methods, including mass spectrometry analyses or complementary technologies such as lectin arrays (17) or glycoprotein arrays (22). It also will be valuable to determine whether increases in certain structures are due to the glycosylation of an increased number of glycan sites, a possibility suggested by a previous study of MUC1 (45), or a shift in structures on the same number of sites.…”

“…Glycan levels can be probed directly from biological samples, and many samples or detection conditions can be processed efficiently in a low volume, high throughput format (16). This method is complementary to lectin microarrays (17)(18)(19), which are useful for measuring glycan levels on individual, purified proteins; glycan microarrays (20,21), which are used to measure the recognition of carbohydrate structures by various glycanbinding reagents; and glycoprotein arrays (22) for examining glycosylation on proteins isolated from biological samples.…”

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

“…In the further pursuit of alternative molecular markers, the patterns of glycosylation have been studied in sera of patients with pancreatic cancer using antibody-and lectin-based sensors (7,8). A similar approach has been used to study pancreatic cyst fluids, targeting representative glycans on prominent candidate glycoprotein markers, including several of the mucins and carcinoembryonic antigen-related cell adhesion molecules.…”

Challenges to Developing Proteomic-Based Breast Cancer Diagnostics

“…The most common approaches for characterizing the protein constituents of N-linked glycoproteins is to use some combination of the following: peptides are generated by digestion with trypsin, the glycopeptides are isolated with one or more lectins or hydrazine linked to a support resin, the bound peptides are treated with protein N-Glycanase F (PNGaseF) to release glycans, then sequencing of the peptides is done by tandem mass spectrometry (Liu et al, 2005;Wang et al, 2006;Zhang et al, 2003;Zielinska et al, 2010). New strategies that probe different lectins bound on multiple array platforms are also emerging (Chen and Haab, 2009;Kuno et al, 2008;Mao et al, 2004;Zhao et al, 2007).…”