title: Hexapeptide library as a universal tool for sample preparation in protein glycosylation analysis.
abstract: Recent analytical advancements allow for large-scale glycomics and glycan-biomarker research with N-glycans released from complex protein mixtures of e.g. plasma with a wide range of protein concentrations. Protein enrichment techniques to obtain samples with a better representation of low-abundance proteins are hardy applied. In this study, hexapeptide ligands previously described for enrichment of low-abundance proteins in proteomics are evaluated for glycan analysis. A repeatable on-bead glycan release strategy was developed, and glycans were analyzed using capillary sieving electrophoresis on a DNA analyzer. Binding of proteins to the hexapeptide library occurred via the protein backbone. At neutral pH no discrimination between protein glycoforms was observed. Interestingly, glycan profiles of plasma with and without hexapeptide library enrichment revealed very similar patterns, despite the vast changes in protein concentrations in the samples. The most significant differences in glycosylation profiles were ascribed to a reduction in immunoglobulin-derived glycans. These results suggest that specific and sensitive biomarkers will be hard to access on the full plasma level using protein enrichment in combination with glycan analysis. Instead, fractionation techniques or profiling strategies on the glycopeptide level after enrichment are proposed for in-depth glycoproteomics research.
authors.affiliation: Forschungszentrum Jülich, Central Division of Analytical Chemistry, Jülich, Germany. c.huhn@fz-juelich.de
authors.forename: Carolin
authors.initials: C
authors.lastname: Huhn
authors.forename: L Renee
authors.initials: LR
authors.lastname: Ruhaak
authors.forename: Manfred
authors.initials: M
authors.lastname: Wuhrer
authors.forename: André M
authors.initials: AM
authors.lastname: Deelder
mesh_terms: D001798:Blood Proteins / Q000032:analysis*; D006031:Glycosylation; D006801:Humans; D009842:Oligopeptides / Q000138:chemical synthesis / Q000737:chemistry*; D019151:Peptide Library; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry; D040901:Proteomics / Q000379:methods*
chemical_list: D001798:Blood Proteins; D009842:Oligopeptides; D019151:Peptide Library; D011134:Polysaccharides