This proposal aims to detect hepatocellular carcinoma (HCC) at a treatable stage by non-invasive methods. To this end, we propose to quantify site specific glycoforms of liver secreted N-glycoproteins (LNP) associated with the progression of liver disease to HCC. We and others have shown that changes in protein glycosylation accompany the development of HCC. Major re-distribution of protein N-glycoforms occurs at the premalignant stage of liver cirrhosis but our latest studies show that specific glycoforms of a glycopeptide of haptoglobin increase exclusively in HCC. We demonstrate that these minor glycoforms of haptoglobin are detectable in serum and have the potential to improve detection of HCC compared to the serologic gold standard, alpha fetoprotein (AFP). This supports our hypothesis that minor site specific glycoforms of abundant liver secreted N-glycoproteins provide HCC specific marker candidates detectable in the circulation. We propose to complete identification of the HCC specific N-glycans;we have already evidence that the glycans are characterized by multiple fucosylations and increased branching. We propose to identify glycopeptides of additional proteins carrying these HCC specific N-glycan modifications by newly optimized mass spectrometric methods. This is done because haptoglobin is downregulated in a fraction of cirrhotics and other proteins may be needed for optimal detection. We expand our informatic analysis to classify all polymorphic and mutant human N- glycoproteins with newly created or abolished N-glycosylation sites. These protein variants are over- represented in the human genome, represent prime candidates for association with diseases, and we expect that they have direct impact on carcinogenesis. We believe that a publically available resource of these variant proteins will stimulate glycoproteomic cancer prevention research. The proposed examination of HCC specific N-glycoforms is feasible because we have already created a repository of samples of HCC patients and cirrhotic controls. This repository, and additional outstanding QC resources, is essential for the isolation of the HCC specific minor glycoforms of liver secreted N-glycoproteins;these glycoforms are not detectable in disease free subjects. By the end of the study, we will have quantified the selected site specific protein glycoforms by targeted LC-MS selective reaction monitoring (SRM) methods. These methods will quantify the HCC associated site specific glycopeptides in serum with an unprecedented accuracy. We do this because we believe that quantification of specific glycoforms of specific glycoprotein peptides offers the highest diagnostic accuracy. Defining clinically applicable cancer markers has potentially far-reaching consequences for disease management and patient health. Our study is expected to generate new hypotheses on the functional impact of protein glycosylation on the development of cancer and to stimulate an entirely new line of cancer prevention and detection research.

Public Health Relevance

The incidence of liver cancer in the United States is steadily increasing. Early detection of cancer improves patient survival. Our study seeks to identify new biomarkers for early detection of liver cancer, which would have a direct impact on the disease management and outcome.

Agency
National Institute of Health (NIH)
Type
Research Project--Cooperative Agreements (U01)
Project #
5U01CA168926-03
Application #
8725604
Study Section
Special Emphasis Panel (ZCA1)
Program Officer
Rinaudo, Jo Ann S
Project Start
Project End
Budget Start
Budget End
Support Year
3
Fiscal Year
2014
Total Cost
Indirect Cost
Name
Georgetown University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
City
Washington
State
DC
Country
United States
Zip Code
20057
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Benicky, Julius; Sanda, Miloslav; Pompach, Petr et al. (2014) Quantification of fucosylated hemopexin and complement factor H in plasma of patients with liver disease. Anal Chem 86:10716-23
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Chandler, Kevin Brown; Brnakova, Zuzana; Sanda, Miloslav et al. (2014) Site-specific glycan microheterogeneity of inter-alpha-trypsin inhibitor heavy chain H4. J Proteome Res 13:3314-29
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Karagiannis, Konstantinos; Simonyan, Vahan; Mazumder, Raja (2013) SNVDis: a proteome-wide analysis service for evaluating nsSNVs in protein functional sites and pathways. Genomics Proteomics Bioinformatics 11:122-6
Pompach, Petr; Brnakova, Zuzana; Sanda, Miloslav et al. (2013) Site-specific glycoforms of haptoglobin in liver cirrhosis and hepatocellular carcinoma. Mol Cell Proteomics 12:1281-93
Sanda, Miloslav; Pompach, Petr; Benicky, Julius et al. (2013) LC-MS3 quantification of O-glycopeptides in human serum. Electrophoresis 34:2342-9
Sanda, Miloslav; Pompach, Petr; Brnakova, Zuzana et al. (2013) Quantitative liquid chromatography-mass spectrometry-multiple reaction monitoring (LC-MS-MRM) analysis of site-specific glycoforms of haptoglobin in liver disease. Mol Cell Proteomics 12:1294-305
Lam, Phuc Vinh Nguyen; Goldman, Radoslav; Karagiannis, Konstantinos et al. (2013) Structure-based comparative analysis and prediction of N-linked glycosylation sites in evolutionarily distant eukaryotes. Genomics Proteomics Bioinformatics 11:96-104

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