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 highthroughput 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.

Serum Galectin-2, -4, and -8 Are Greatly Increased in Colon and Breast Cancer Patients and Promote Cancer Cell Adhesion to Blood Vascular Endothelium

“…MUC1 peptides were synthesized, O-glycosylated in vitro using various recombinant glycosyltransferases and purified exactly as previously described (23,24). The glycopeptides were printed on Schott Nexterion Slide H (Schott AG) on a BioRobotics MicroGrid II spotter (Genomics Solution) with a 0.21-mm pitch using Stealth 3B Micro Spotting Pins (Telechem International ArrayIt Division).…”

A Contra Capture Protein Array Platform for Studying Post-translationally Modified (PTM) Auto-antigenomes

“…These included: (1) a 60-mer MUC1-TR comprising 3 repeats of the 20 AA tandem repeat (PDTRPAPGSTAPPAHGVTSA) of MUC1; (2) an NRP2 fragment (SKPTVETLGPTVKSEETTTP); (3) a POMC fragment (PGNGDEQ-PLTENPRKYVMGHFR); (4) a CD55 (SRTTKHFHETTPNKGSGTTS) fragment, (5) a wild type APOE fragment (VEQGRVRAATVGSLAGQP), and (6) a mutant APOE fragment (VEQGRVRAAAVGALAGQ) with the glycosylation site Thr and Ser mutated to Ala. Tn glycosylation was performed using GalNac-T2, T3 or T11 (kindly provided by Dr. Henrik Clausen) with Uridine 5Ј-diphospho-N-acetylgalactosamine disodium salt (U5252 Sigma) (2 mM) and MnCl2 (Sigma) (10 mM) in glycosylation buffer (25 mM TRIS (MP Biomedicals, Santa Ana, CA), pH7.5, 10 mM MnCl2, 0.25% Triton X-100 (Sigma) at 37°C for 18 h (22,44).…”

“…In addition to the challenges associated with proteome level purification of full-length human proteins, it is often impractical to modify thousands of individual recombinant proteins to print conventional protein arrays (24). Printed proteins are usually immobilized on array substrates through nonspecific hydrophobic/electrostatic interactions, which do not survive modification protocols that often include extended incubation at high temperatures (11,22). In addition, the exposed amine groups of the lysine residues are prone to reactivity and may result in steric hindrance that mask reactive epitopes.…”

Characterization of the Viral O -Glycopeptidome: a Novel Tool of Relevance for Vaccine Design and Serodiagnosis

“…Peptides were prepared by automated peptide synthesis on a Syro II peptide synthesizer (MultiSynTech, Witten, Germany) by a modified 9-fluorenylmethoxy carbonyl (Fmoc)-solidphase peptide synthesis (SPPS) methodology protocol (see the supple-mental Methods in the supplemental material for details) (5). Helix pomatia agglutinin (HPA)-purified mgG-2 was prepared as previously described (37 After printing, the slides were incubated at 70% humidity for 60 min, and the remaining NHS groups were deactivated in blocking buffer (50 mM ethanolamine in 50 mM borate buffer; pH 9.2) for 1 h and then rinsed in Millipore water and spun dry.…”