Hepatocellular carcinoma (HCC) is the third most common cause of cancer-related death worldwide and is rising in incidence in the US. 90% of patients in the US with liver cancer have underlying cirrhosis, thus guideline recommendations recommend surveillance in all patients with cirrhosis to facilitate early detection. Unfortunately, only 20-30% of patients are detected with early detection and are thus eligible for potentially curative treatments. There is an unmet need for reliable biomarkers for HCC to facilitate adherence to screening and for early detection. In the proposed work we will develop early detection strategies for HCC based on glycoproteomic profiles. Unique changes in glycosylation in proteins, which involve structural changes in glycan groups, have been shown to be important serum biomarkers for early cancer detection. Importantly, the subtle changes may only involve minor structures but they can be very specific in differentiating cirrhosis versus early versus late stage HCC. In addition, these changes may be specific to the etiology of liver disease. These glycan structural changes will be detected and monitored quantitatively using a mass spectrometry approach which has proven to be an accurate way to characterize even minor changes in structure which may be significant as biomarkers based on our previous mass analysis, tandem mass spectrometry measurements and databases which have been developed for glycan and glycopeptide analysis. This will be demonstrated for both glycan and glycopeptide screening from serum using novel extraction and separation methods coupled to mass spectrometry which can ultimately be used to distinguish early stage HCC from cirrhosis. The proposed work will deliver separations and mass spec methods enabling isomeric separation of glycans and glycopeptides, permitting unequivocal assignment of protein glycosylation related to disease state. We will be able to distinguish different isomeric forms of fucosylation and sialylation which may contain important disease related markers. Novel software will be developed and used to assign these glycan structures. The markers will be discovered for specific etiologies of HCV-related, alcohol-related and NAFLD-related etiologies of HCC. This will be a multisite study to include all components required for a tumor biomarker lab including samples and sample preparation, separations and mass spec analysis, bioinformatics evaluation and statistical analysis. Ultimately, we will develop methods for discovery of glycan/glycopeptide markers from patient serum, the identification of potential markers and the development of new assays to provide a limited confirmation of these markers.