Diethylaminoethyl Sepharose (DEAE-Sepharose) microcolumn for enrichment of glycopeptides.
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.
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
He
H
Zhu
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
Xu
X
Li
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
Jingyao
J
Qu
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
Cong
C
Xiao
State Key Laboratory of Medicinal Chemical Biology and College of Pharmacy, Nankai University, Haihe Education Park, 38 Tongyan Road, Tianjin, 300353, China.
Kuan
K
Jiang
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
Ebtesam
E
Gashash
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
Ding
D
Liu
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA.
Jing
J
Song
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.
Jiansong
J
Cheng
Department of Chemistry and Center for Diagnostics & Therapeutics, Georgia State University, Atlanta, GA, 30303, USA. cma@gsu.edu.
Cheng
C
Ma
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.
Peng George
PG
Wang
D006020:Glycopeptides / Q000097:blood / Q000737:chemistry* / Q000302:isolation & purification*; D006031:Glycosylation; D006801:Humans; D012685:Sepharose / Q000737:chemistry*; D052616:Solid Phase Extraction / Q000295:instrumentation*
D006020:Glycopeptides; D012685:Sepharose
Amino acids/peptides; Biological samples; Mass spectrometry/ICP-MS