Comparison of three glycoproteomic methods for the analysis of the secretome of CHO cells treated with 1,3,4-O-Bu3 ManNAc

Joseph L. Mertz, Shisheng Sun, Bojiao Yin, Yingwei Hu, Rahul Bhattacharya, Michael J. Bettenbaugh, Kevin J. Yarema, Hui Zhang

Research output: Contribution to journalArticlepeer-review


Comprehensive analysis of the glycoproteome is critical due to the importance of glycosylation to many aspects of protein function. The tremendous complexity of this post-translational modification, however, makes it difficult to adequately characterize the glycoproteome using any single method. To overcome this pitfall, in this report we compared three glycoproteomic analysis methods; first the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, second, solid-phase extraction of N-linked glycoproteins (SPEG), and third, hydrophilic interaction liquid chromatography (HILIC) by characterizing N-linked glycosites in the secretome of Chinese hamster ovary (CHO) cells. Interestingly, the glycosites identified by SPEG and HILIC overlapped considerably whereas NGAG identified many glycosites not observed in the other two methods. Further, utilizing enhanced intact glycopeptide identification afforded by the NGAG workflow, we found that the sugar analog 1,3,4-O-Bu3 ManNAc, a “high flux” metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).

Original languageEnglish (US)
Article number144
Pages (from-to)1-16
Number of pages16
Issue number4
StatePublished - Dec 2020


  • 1,3,4-O-Bu ManNAc
  • CHO
  • Glycoproteomics
  • Sialylation

ASJC Scopus subject areas

  • Bioengineering

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