Analytical and Bioanalytical Chemistry

, Volume 409, Issue 12, pp 3077–3087 | Cite as

Sequential fragment ion filtering and endoglycosidase-assisted identification of intact glycopeptides

  • Zixiang Yu
  • Xinyuan Zhao
  • Fang Tian
  • Yang Zhao
  • Yong Zhang
  • Yi Huang
  • Xiaohong Qian
  • Wantao YingEmail author
Research Paper


Detailed characterization of glycoprotein structures requires determining both the sites of glycosylation as well as the glycan structures associated with each site. In this work, we developed an analytical strategy for characterization of intact N-glycopeptides in complex proteome samples. In the first step, tryptic glycopeptides were enriched using ZIC-HILIC. Secondly, a portion of the glycopeptides was treated with endoglycosidase H (Endo H) to remove high-mannose (Man) and hybrid N-linked glycans. Thirdly, a fraction of the Endo H-treated glycopeptides was further subjected to PNGase F treatment in 18O water to remove the remaining complex glycans. The intact glycopeptides and deglycosylated peptides were analyzed by nano-RPLC–MS/MS, and the glycan structures and the peptide sequences were identified by using the Byonic or pFind tools. Sequential digestion by endoglycosidase provided candidate glycosites information and indication of the glycoforms on each glycopeptide, thus helping to confine the database search space and improve the confidence regarding intact glycopeptide identification. We demonstrated the effectiveness of this approach using RNase B and IgG and applied this sequential digestion strategy for the identification of glycopeptides from the HepG2 cell line. We identified 4514 intact glycopeptides coming from 947 glycosites and 1011 unique peptide sequences from HepG2 cells. The intensity of different glycoforms at a specific glycosite was obtained to reach the occupancy ratios of site-specific glycoforms. These results indicate that our method can be used for characterizing site-specific protein glycosylation in complex samples.

Graphical abstract

Through integrating the information of intact glycopeptide, fragment ions filters and endoglycosidase digestion, the reliability of the identification could be significantly improved. We quantified the site-specific glycoforms occupancy ratios through the MS response signaling of each glycopeptide at the same time.


Glycoprotein Glycosylation HILIC Mass spectrometry Byonic 



We are grateful for the financial support from the National Key Program for Basic Research of China (2016YFA0501300, 2014CBA02001), the State Key Laboratory of NBC Protection of Civilian (SKLNBC03010), and the National Natural Science Foundation of China (81530021).

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Supplementary material

216_2017_195_MOESM1_ESM.pdf (109 kb)
ESM 1 (PDF 109 kb)
216_2017_195_MOESM2_ESM.xlsx (458 kb)
ESM 2 (XLSX 457 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2017

Authors and Affiliations

  • Zixiang Yu
    • 1
    • 2
  • Xinyuan Zhao
    • 2
  • Fang Tian
    • 2
  • Yang Zhao
    • 2
  • Yong Zhang
    • 2
  • Yi Huang
    • 2
  • Xiaohong Qian
    • 2
  • Wantao Ying
    • 1
    • 2
    Email author
  1. 1.Anhui Medical UniversityHefeiChina
  2. 2.National Center for Protein Sciences Beijing, State Key Laboratory of Proteomics, Beijing Proteome Research CenterBeijing Institute of Radiation MedicineBeijingChina

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