title: Application of a strong anion exchange material in electrostatic repulsion-hydrophilic interaction chromatography for selective enrichment of glycopeptides.
abstract: Glycoproteins are involved in various cellular activities, including inter- and extracellular signaling. However, glycopeptide signals are significantly suppressed by coeluting non-glycosylated peptides in mass spectrometry-based analysis. For detailed elucidation of the biological functions of glycoproteins, selective enrichment of glycopeptides from non-glycosylated peptides is crucial. In the present study, a SAX material, XCharge SAX, was used in a column in the ERLIC mode with the aim of specifically enriching glycopeptides. Enrichment conditions were initially optimized, and selectivity, glycosylation heterogeneity coverage and detection sensitivity of XCharge SAX were subsequently assessed. In the selectivity assessment, glycopeptides were effectively isolated from a peptide mixture (human serum immunoglobulin G (IgG) and human serum albumin digests) and a tryptic digest of human serum using XCharge SAX. In the evaluation of glycosylation heterogeneity coverage, five glycosites and eleven glycopeptides from horseradish peroxidase were identified after enrichment with XCharge SAX. In detection sensitivity assessment, glycopeptides within four orders of magnitude were identified after enrichment with XCharge SAX. In addition, volatile solvents were used in the loading and eluting buffers so that desalting was not necessary for ERLIC fractions. Our results collectively support the utility of XCharge SAX as a suitable chromatographic material for global glycosylation site analysis.
authors.affiliation: Dalian Institute of Chemical Physics, Chinese Academy of Sciences, Dalian 116023, China.
authors.forename: Liwei
authors.initials: L
authors.lastname: Cao
authors.forename: Long
authors.initials: L
authors.lastname: Yu
authors.forename: Zhimou
authors.initials: Z
authors.lastname: Guo
authors.forename: Xiuling
authors.initials: X
authors.lastname: Li
authors.forename: Xinya
authors.initials: X
authors.lastname: Xue
authors.forename: Xinmiao
authors.initials: X
authors.lastname: Liang
mesh_terms: D000837:Anion Exchange Resins / Q000737:chemistry*; D002845:Chromatography / Q000379:methods*; D006020:Glycopeptides / Q000737:chemistry* / Q000302:isolation & purification; D057927:Hydrophobic and Hydrophilic Interactions; D055672:Static Electricity
chemical_list: D000837:Anion Exchange Resins; D006020:Glycopeptides