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There is increasing evidence that glycosylation is highly sensitive to the biochemical environment and it plays a significant role in several important biological processes including transcription, differentiation, and signaling, as well as oncogenesis and metastasis. Therefore the tumor specific alterations in protein glycosylation on the cell surface and in body fluids might be potential targets for the development of new cancer diagnostics and therapeutics. The recent progress in various proteomic technologies allows high throughput screening of protein-based biomarkers from patients’ serum. However, only a limited number of proteins can be detected by current methods such as shotgun-proteomics, mainly because of the enormous concentration distribution of the serum proteome. To overcome this difficulty and isolate potential cancer-specific biomarkers for diagnosis and treatment, we established a new glycoproteomic screening system involving the following steps: (1) immunodepletion of highly abundant proteins, (2) targeted enrichment of glycoproteins by lectin column chromatography, and (3) a quantitative proteome analysis using 12C6- or 13C6-NBS (2-nitrobenzensulfenyl) stable isotope labeling followed by MALDI-QIT-TOF mass spectrometric analysis. This methodology made possible a highly sensitive and quantitative screening for carbohydrate-targeting serum tumor markers, which could identify low and high abundant proteins (102 ~ 108 pg/ml in serum). To date, we have identified 39 serum glycoproteins as candidate biomarkers indicating that there is more than a 2-fold difference in the α1,6-fucosylation level between serum samples from five lung adenocarcinoma patients and a mixture of samples from five healthy individuals (control). In addition, we validated the data by means of immunoprecipitation and lectin blot analysis (IP-lectin blotting) of two candidate serum proteins, haptoglobin and pigment epithelial derived factor (PEDF), on which increased level of α1,6-fucosylation was detected in many of the lung cancer samples. The results of IP-lectin blotting analyses using 8 serum samples derived from lung cancer patients and 8 from healthy volunteers, exactly matched the quantification data obtained by NBS-based MS screening. This new approach will provide a wide range of applications with rapid and reproducible profiling of cancer-specific alterations of glycan structures on multiple serum proteins, which should be valuable for biomarker development and the study of variations in glycan structures in any type of human disease.

98th AACR Annual Meeting-- Apr 14-18, 2007; Los Angeles, CA