A High-Throughput O-Glycopeptide Discovery Platform for Seromic Profiling

Abstract

Biomarker microarrays are becoming valuable tools for serological screening of disease-associated autoantibodies. Post-translational modifications (PTMs) such as glycosylation extend the range of protein function, and a variety of glycosylated proteins are known to be altered in disease progression. Here, we have developed a synthetic screening microarray platform for facile display of O-glycosylated peptides (O-PTMs). By introducing a capping step during chemical solid-phase glycopeptide synthesis, selective enrichment of N-terminal glycopeptide end products were achieved on an amine-reactive hydrogel coated microarray glass surface, allowing high-throughput display of large numbers of glycopeptides. Utilizing a repertoire of recombinant glycosyltransferases enabled further diversification of the array libraries in-situ and display of a new level of potential biomarker candidates for serological screening. As proof-of-concept we have demonstrated that MUC1 glycopeptides could be assembled and used to detect autoantibodies in vaccine induced disease free breast cancer patients and in patients, with confirmed disease at time of diagnosis.

Synthetic Antitumor Vaccines Containing MUC1 Glycopeptides with Two Immunodominant Domains—Induction of a Strong Immune Response against Breast Tumor Tissues

“…It also differs from those of autoantibodies isolated from serum of tumor patients. [12] These divergent recognition selectivities probably can be traced back to different glycosylation positions in the MUC1 tandem repeat sequence 1:…”

“…The MUC1 tetanus toxoid vaccines [9] described above carried the T antigen or the sialyl-T N antigen side chains at threonine-6 of 1, whereas the SM3 antibody [10,11] as well as the autoantibodies in the sera of patients [12] showed intensive binding to MUC1 glycopeptides glycosylated in the GSTA region (Ser 17 , Thr 18 ). NMR spectroscopic analyses had shown that glycan side chains in the STAPPA peptide sequence of MUC1 influence the conformation of this peptide segment.…”

Deciphering Enzyme Function Using Peptide Arrays

“…or in release of a part of the immobilised peptide derivative (proteases, phosphatases, histone deacetylases and lysine demethylases, etc.). Studies with kinases, phosphatases, proteases, (comprehensively reviewed in Table 1 in supplementary material), lysine methyl-transferases [117,118], arginine methyl-transferases [119], histone deacetylases [120][121][122][123], peptidyl-prolyl-cis/trans-isomerases [75,[124][125][126][127], glycosyltransferases [128][129][130], ADP-ribosyl-transferases [131,132], hydrolases [133], esterases [134] and ubiquitin-or SUMOligases [135][136][137] have been described using peptide arrays on cellulose membranes or peptide microarrays on glass slides. This review will summarise applications of peptide (micro)arrays for kinase, phosphatase and protease research in more detail.…”

Site-Specific Modification of Recombinant Proteins: A Novel Platform for Modifying Glycoproteins Expressed in E. coli

“…To determine recognition sequence requirements for individual transferases, in vitro reactions involving purified enzymes and substrates are typically used 31À39 (a pH-based high-throughput assay for glycosyltransferase activity has been described, but it has not yet been used for the characterization of GalNAc-T isoforms 40 ). Recently, the in vitro use of glycosyltransferases has been extended to engineering applications such as the generation of glycopeptide libraries 41 and use in bioconjugate chemistry. 42 To find a suitable OGRS for hGalNAc-T2, we evaluated a small subset of the OGRS present in O-GlycBase v 6.00 for use as glycosylation targets on recombinant proteins.…”