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

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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 Fe3O4 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.

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Abbreviations

Fuc:

Fucose

Gal:

Galactose

GlcNAc:

N-Acetylglucosamine

Hex:

Hexose

HexNAc:

N-Acetylhexosamine

HILIC:

Hydrophilic interaction liquid chromatography

MS:

Mass spectrometry

Neu5Ac:

N-Acetylneuraminic acid

ULF:

Uterine luminal fluid

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Acknowledgments

This work was supported by Taiwan NSC grants 94-3112-B-001-009-Y and 95-3112-B-001-014 to the NRPGM Core Facilities for Proteomics located at the Institute of Biological Chemistry, Academia Sinica. The LTQ-Orbitrap data were additionally acquired at the Academia Sinica common mass spectrometry facilities, also at the Institute of Biological Chemistry. We are grateful to Dr. Sin-Tak Chu (Institute of Biochemical Sciences, National Taiwan University) for providing the mouse uterine luminal fluid samples.

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  1. NRPGM Core Facilities for Proteomics and Institute of Biological Chemistry, Academia Sinica, 128, Academia Road, Sec 2, Nankang, Taipei, 11529, Taiwan

    Chu-Wei Kuo, I-Lin Wu, He-Hsuan Hsiao & Kay-Hooi Khoo

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  1. Chu-Wei Kuo
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  2. I-Lin Wu
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Correspondence to Kay-Hooi Khoo.

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Kuo, CW., Wu, IL., Hsiao, HH. et al. Rapid glycopeptide enrichment and N-glycosylation site mapping strategies based on amine-functionalized magnetic nanoparticles. Anal Bioanal Chem 402, 2765–2776 (2012). https://doi.org/10.1007/s00216-012-5724-1

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  • DOI: https://doi.org/10.1007/s00216-012-5724-1

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