Elsevier

Analytical Biochemistry

Volume 348, Issue 2, 15 January 2006, Pages 259-268
Analytical Biochemistry

Site-specific N-glycosylation analysis of human plasma ceruloplasmin using liquid chromatography with electrospray ionization tandem mass spectrometry

https://doi.org/10.1016/j.ab.2005.10.036Get rights and content

Abstract

Ceruloplasmin has ferroxidase activity and plays an essential role in iron metabolism. In this study, a site-specific glycosylation analysis of human ceruloplasmin (CP) was carried out using reversed-phase high-performance liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS). A tryptic digest of carboxymethylated CP was subjected to LC-ESI-MS/MS. Product ion spectra acquired data-dependently were used for both distinction of the glycopeptides from the peptides using the carbohydrate B-ions, such as m/z 204 (HexNAc) and m/z 366 (HexHexNAc), and identification of the peptide moiety of the glycopeptide based on the presence of the b- and y-series ions derived from the peptide. Oligosaccharide composition was deduced from the molecular weight calculated from the observed mass of the glycopeptide and theoretical mass of the peptide. Of the seven potential N-glycosylation sites, four (Asn119, Asn339, Asn378, and Asn743) were occupied by a sialylated biantennary or triantennary oligosaccharide with fucose residues (0, 1, or 2). A small amount of sialylated tetraantennary oligosaccharide was detected. Exoglycosidase digestion suggested that fucose residues were linked to reducing end GlcNAc in biantennary oligosaccharides and to reducing end and/or α1–3 to outer arms GlcNAc in triantennary oligosaccharides and that roughly one of the antennas in triantennary oligosaccharides was α2–3 sialylated and occasionally α1–3 fucosylated at GlcNAc.

Section snippets

Materials

Acetonitrile, formic acid, and guanidine hydrochloride were purchased from Wako Pure Chemicals Industries (Osaka, Japan). Purified human CP was purchased from Calbiochem (San Diego, CA, USA). Modified trypsin was purchased from Promega (Madison, WI, USA). α2–3 Neuraminidase (EC 3.2.1.18) of Macrobdella decora, a recombinant form, and α1–3,4 fucosidase (EC 3.2.1.51) from Xanthomonas sp. were purchased from Calbiochem. α2–3,6,8,9 Neuraminidase (EC 3.2.1.18) of Arthrobacter ureafaciens, a

Peptide mapping of tryptic digest of human CP (LC-ESI-MS/MS in m/z range of 400–2000)

The amino acid sequence of human CP (National Center for Biotechnology Information protein database: P00450) is shown in Fig. 1. The tryptic peptides, including potential N-glycosylation sites, are shown in bold type. Trypsin can digest human CP into seven glycopeptides containing only one potential N-glycosylation site. To determine the glycosylation state at each glycosylation site, we performed mass spectrometric peptide mapping of the tryptic digest of CP. An aliquot of 0.2 μg of the tryptic

Discussion

A site-specific glycosylation analysis of human CP was conducted using LC-ESI-MS/MS, where product ion spectra were acquired in a data-dependent manner. The collision energy for the product ion scan was adjusted from 30 to 80 eV depending on the size and charge of the precursor ion. Under these conditions, peptide precursor ions were degraded and produced b- and y-series fragment ions derived from the amino acid sequence. Glycopeptide precursor ions produced abundant carbohydrate ions (m/z 204,

Acknowledgments

This study was supported by a Grant-in-Aid for Research on Health Sciences focusing on Drug Innovation from the Japan Health Sciences Foundation.

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