title: Resolving Isomeric Glycopeptide Glycoforms with Hydrophilic Interaction Chromatography (HILIC).
abstract: The ability to resolve glycans while attached to tryptic peptides would greatly facilitate glycoproteomics, as this would enable site-specific glycan characterization. Peptide/glycopeptide separations are typically performed using reversed-phase liquid chromatography (RPLC), where retention is driven by hydrophobic interaction. As the hydrophilic glycans do not interact significantly with the RPLC stationary phase, it is difficult to resolve glycopeptides that differ only in their glycan structure, even when these differences are large. Alternatively, glycans interact extensively with the stationary phases used in hydrophilic interaction chromatography (HILIC), and consequently, differences in glycan structure have profound chromatographic shifts in this chromatographic mode. Here, we evaluate HILIC for the separation of isomeric glycopeptide mixtures that have the same peptide backbone but isomeric glycans. Hydrophilic functional groups on both the peptide and the glycan interact with the HILIC stationary phase, and thus, changes to either of these moieties can alter the chromatographic behavior of a glycopeptide. The interactive processes permit glycopeptides to be resolved from each other based on differences in their amino acid sequences and/or their attached glycans. The separations of glycans in HILIC are sufficient to permit resolution of isomeric N-glycan structures, such as sialylated N-glycan isomers differing in α2-3 and α2-6 linkages, while these glycans remain attached to peptides.
authors: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA;
authors: Yining
authors: Y
authors: Huang
authors: College of Life Science, Shandong Agricultural University, Taian, Shandong Province, P.R. China; and.
authors: Yongxin
authors: Y
authors: Nie
authors: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA;; Advanced Materials Technology, Incorporated, Wilmington, Delaware 19810, USA.
authors: Barry
authors: B
authors: Boyes
authors: Complex Carbohydrate Research Center, University of Georgia, Athens, Georgia 30602, USA;
authors: Ron
authors: R
authors: Orlando
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002417:Cattle; D002850:Chromatography, Gel; D056148:Chromatography, Reverse-Phase; D060748:Fetuins / Q000737:chemistry / Q000302:isolation & purification*; D006020:Glycopeptides / Q000737:chemistry / Q000302:isolation & purification*; D006801:Humans; D057927:Hydrophobic and Hydrophilic Interactions; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification; D011485:Protein Binding; D012680:Sensitivity and Specificity
chemical_list: D060748:Fetuins; D006020:Glycopeptides; D011134:Polysaccharides
keywords: Fetuin; HILIC; IgGs; Isomeric Separation