Profiling cancer biomarkers through metabolic oligosaccharide engineering and glycoproteomics

Of the proteins approved by the Food and Drug Administration for use as biomarkers of cancer, the majority are post-translationally modified with glycans. Changes in glycan quantity and structure are associated with malignancy. However, glycosylation is not a DNA template-driven process; consequently, commonly used techniques in molecular biology are not adequate for glycoprotein analysis. The development of chemical tools for glycoprotein analysis has shown great promise for the identification of new biomarkers.;In Chapter 1, the techniques currently used to enrich and identify glycoproteins are surveyed. Cell-surface protein modification is highlighted in particular, as this class of proteins represents a significant group of cancer targets. Additional discussion of the use of mass spectrometry as an analytical tool for glycoprotein characterization is included. Finally, this chapter introduces the concept of using chemical functionalities to label glycans metabolically for subsequent identification. Chapter 2 describes the application of metabolic labeling to the study of fucosylation, up-regulation of which is associated with aggressive disease. These studies demonstrate that immortalized cancer cell lines will incorporate a fucose analog, 6-azidofucose, into cell-surface glycans.;In addition to labeling fucosylated glycans in cultured cells, the Bertozzi lab has shown that other azidosugars are metabolically incorporated into cell-surface glycoproteins in healthy mice, particularly in well-vascularized organs such as the liver. Chapters 3 and 4 describe efforts to establish the utility of metabolic labeling in various cancer models. Chapter 3 presents studies using a transgenic mouse model of liver cancer. Azidosugars were found to differentially label cancerous versus healthy tissue. In Chapter 4, a method for the specific labeling of cell-surface glycans in prostate cancer cell lines and the subsequent enrichment and identification of these glycoproteins by mass spectrometry is presented and validated. The method was then applied to cultured human prostate tissue slices. Freshly excised human cancer tissues robustly metabolized azidosugars. These results establish a platform for the use of azidosugars in the identification of new glycan cancer biomarkers.