A strategy for discovery of cancer glyco‐biomarkers in serum using newly developed technologies for glycoproteomics

Detection of cancer at early stages that can be treated through surgery is a difficult task. One methodology for cancer biomarker discovery exploits the fact that glycoproteins produced by cancer cells have altered glycan structures, although the proteins themselves are common, ubiquitous, abundant, and familiar. However, as cancer tissue at the early stage probably constitutes less than 1% of the normal tissue in the relevant organ, only 1% of the relevant glycoproteins in the serum should have altered glycan structures. Here, we describe our strategy to approach the detection of these low-level glycoproteins: (a) a quantitative real-time PCR array for glycogenes to predict the glycan structures of secreted glycoproteins; (b) analysis by lectin microarray to select lectins that distinguish cancer-related glycan structures on secreted glycoproteins; and (c) an isotope-coded glycosylation site-specific tagging high-throughput method to identify carrier proteins with the specific lectin epitope. Using this strategy, we have identified many glycoproteins containing glycan structures that are altered in cancer cells. These candidate glycoproteins were immunoprecipitated from serum using commercially available antibodies, and their glycan alteration was examined by a lectin microarray. Finally, they were analyzed by multistage tandem MS.