Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles

• Published in: Analytical and bioanalytical chemistry 2012-03, Vol.402 (9), p.2765-2776
• Main Authors: Kuo, Chu-Wei, Wu, I-Lin, Hsiao, He-Hsuan, Khoo, Kay-Hooi
• Format: Article
• Language: English
• Subjects: Amines; Analytical Chemistry; Animals; Biochemistry; Characterization and Evaluation of Materials; Chemistry; Chemistry and Materials Science; Chromatography, Liquid; Comparative analysis; Enrichment; Female; Food Science; Glycan; Glycopeptides; Glycopeptides - chemistry; Glycoprotein; Glycoproteins; Glycosylation; Health aspects; Hostages; Ionization; Laboratory Medicine; Liquid chromatography; Magnetics; Mapping; Mass Spectrometry; Mice; Monitoring/Environmental Analysis; Nanoparticles; Nanoparticles - chemistry; Nanostructure; Original Paper; Peptide Mapping - instrumentation; Peptide Mapping - methods; Peptides; Polysaccharides; Proteomics - instrumentation; Proteomics - methods; Strategy
• ISSN: 1618-2642; 1618-2650
• DOI: 10.1007/s00216-012-5724-1

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.