Random Glycopeptide Bead Libraries for Seromic Biomarker Discovery
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- 作者：Stjepan K. Kraun ; Emiliano Cl ; Henrik Clausen ; Steven B. Levery ; Knud J. Jensen ; Ola Blixt
- 刊名：Journal of Proteome Research
- 出版年：2010
- 出版时间：December 3, 2010
- 年：2010
- 卷：9
- 期：12
- 页码：6705-6714
- 全文大小：491K
- 年卷期：v.9,no.12(December 3, 2010)
- ISSN：1535-3907
文摘
Identification of disease-specific biomarkers is important to address early diagnosis and management of disease. Aberrant post-translational modifications (PTM) of proteins such as O-glycosylations (O-PTMs) are emerging as triggers of autoantibodies that can serve as sensitive biomarkers. Here we have developed a random glycopeptide bead library screening platform for detection of autoantibodies and other binding proteins. Libraries were build on biocompatible PEGA beads including a safety-catch C-terminal amide linker (SCAL) that allowed mild cleavage conditions (I2/NaBH4 and TFA) for release of glycopeptides and sequence determination by ESI-Orbitrap-MSn. As proof-of-principle, tumor -specific glycopeptide reporter epitopes were built-in into the libraries and were detected by tumor-specific monoclonal antibodies and autoantibodies from cancer patients. Sequenced and identified glycopeptides were resynthesized at the preparative scale by automated parallel peptide synthesis and printed on microarrays for validation and broader analysis with larger sets of sera. We further showed that chemical synthesis of the monosaccharide O-glycopeptide library (Tn-glycoform) could be diversified to other tumor glycoforms by on-bead enzymatic glycosylation reactions with recombinant glycosyltransferases. Hence, we have developed a high-throughput flexible platform for rapid discovery of O-glycopeptide biomarkers and the method has applicability in other types of assays such as lectin/antibody/enzyme specificity studies as well as investigation of other PTMs.