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Anal Bioanal Chem. 2017 Jan;409(2):511-518. doi: 10.1007/s00216-016-9937-6. Epub 2016 Sep 27.

Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides.

Author information

1
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
2
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, China.
3
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, China. jiansongcheng@nankai.edu.cn.
4
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA. cma@gsu.edu.
5
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, China. pwang@nankai.edu.cn.

Abstract

N-Glycosylation is one of the most prevalent protein post-translational modifications and is involved in many biological processes, such as protein folding, cellular communications, and signaling. Alteration of N-glycosylation is closely related to the pathogenesis of diseases. Thus, the investigation of protein N-glycosylation is crucial for the diagnosis and treatment of disease. In this research, we applied diethylaminoethanol (DEAE) Sepharose solid-phase extraction microcolumns for N-glycopeptide enrichment. This method integrated the advantages of Click Maltose and zwitterionic HILIC (ZIC-HILIC) and showed a relatively higher specificity for N-glycosylated peptides. This strategy was then applied to tryptic digests of normal human serum, followed by deglycosylation using peptide-N-glycosidase F (PNGase F) in H218O. Subsequent LC-MS/MS analysis allowed for the assignment of 219 N-glycosylation sites from 115 serum N-glycoproteins. This study provides an alternative approach for N-glycopeptide enrichment and the method employed is effective for large-scale N-glycosylation site identification. Graphical abstract Proposed mechanism of glycopeptides enrichment using DEAE-Sepharose.

KEYWORDS:

Amino acids/peptides; Biological samples; Mass spectrometry/ICP-MS

PMID:
27679458
DOI:
10.1007/s00216-016-9937-6
[Indexed for MEDLINE]

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