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