title: Random glycopeptide bead libraries for seromic biomarker discovery.
abstract: 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 (I(2)/NaBH(4) and TFA) for release of glycopeptides and sequence determination by ESI-Orbitrap-MS(n). 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.
authors.affiliation: Department of Cellular and Molecular Medicine, University of Copenhagen, Copenhagen Center for Glycomics, Blegdamsvej 3b, DK-2200 Copenhagen N, Denmark.
authors.forename: Stjepan K
authors.initials: SK
authors.lastname: Kracun
authors.forename: Emiliano
authors.initials: E
authors.lastname: Cló
authors.forename: Henrik
authors.initials: H
authors.lastname: Clausen
authors.forename: Steven B
authors.initials: SB
authors.lastname: Levery
authors.forename: Knud J
authors.initials: KJ
authors.lastname: Jensen
authors.forename: Ola
authors.initials: O
authors.lastname: Blixt
mesh_terms: D000595:Amino Acid Sequence; D000911:Antibodies, Monoclonal / Q000276:immunology; D001323:Autoantibodies / Q000097:blood / Q000276:immunology; D014408:Biomarkers, Tumor / Q000032:analysis* / Q000097:blood / Q000276:immunology; D006020:Glycopeptides / Q000032:analysis* / Q000138:chemical synthesis / Q000276:immunology; D006031:Glycosylation; D006801:Humans; D008969:Molecular Sequence Data; D018396:Mucin-1 / Q000032:analysis* / Q000097:blood / Q000276:immunology; D009369:Neoplasms / Q000097:blood; D019151:Peptide Library; D011506:Proteins / Q000032:analysis / Q000276:immunology / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000911:Antibodies, Monoclonal; D001323:Autoantibodies; D014408:Biomarkers, Tumor; D006020:Glycopeptides; D018396:Mucin-1; D019151:Peptide Library; D011506:Proteins