title: Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles.
abstract: Glycoproteins secreted or expressed on the cell surface at specific pathophysiological stages are well-recognized disease biomarkers and therapeutic targets. While mapping of specific glycan structures can be performed at the level of released glycans, site-specific glycosylation and identification of specific protein carriers can only be determined by analysis of glycopeptides. A key enabling step in mass spectrometry (MS)-based glycoproteomics is the ability to selectively or non-selectively enrich for the glycopeptides from a total pool of a digested proteome for MS analysis since the highly heterogeneous glycopeptides are usually present at low abundance and ionize poorly compared with non-glycosylated peptides. Among the most common approaches for non-destructive and non-glycan-selective glycopeptide enrichment are strategies based on various forms of hydrophilic interaction liquid chromatography (HILIC). We present here a variation of this method using amine-derivatized Fe(3)O(4) nanoparticles, in concert with in situ peptide N-glycosidase F digestion for direct matrix-assisted laser desorption/ionization–mass spectrometry analysis of N-glycosylation sites and the released glycans. Conditions were also optimized for efficient elution of the enriched glycopeptides from the nanoparticles for on-line nanoflow liquid chromatography–MS/MS analysis. Successful applications to single glycoproteins as well as total proteomic mixtures derived from biological fluids established the unrivaled practical versatility of this method, with enrichment efficiency comparable to other HILIC-based methods.
authors.affiliation: NRPGM Core Facilities for Proteomics, Academia Sinica, Nankang, Taipei, Taiwan.
authors.forename: Chu-Wei
authors.initials: CW
authors.lastname: Kuo
authors.forename: I-Lin
authors.initials: IL
authors.lastname: Wu
authors.forename: He-Hsuan
authors.initials: HH
authors.lastname: Hsiao
authors.forename: Kay-Hooi
authors.initials: KH
authors.lastname: Khoo
mesh_terms: D000818:Animals; D002853:Chromatography, Liquid; D005260:Female; D006020:Glycopeptides / Q000737:chemistry*; D006031:Glycosylation; D008280:Magnetics; D013058:Mass Spectrometry; D051379:Mice; D053758:Nanoparticles / Q000737:chemistry*; D010449:Peptide Mapping / Q000295:instrumentation / Q000379:methods*; D040901:Proteomics / Q000295:instrumentation / Q000379:methods*
chemical_list: D006020:Glycopeptides