Comparison of Three Glycoproteomic Methods for the Analysis of the Secretome of CHO Cells Treated with 1,3,4-O-Bu3ManNAc

Abstract

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-Bu3ManNAc, a “high flux” metabolic precursor for sialic acid biosynthesis, increased sialylation of secreted proteins including recombinant human erythropoietin (rhEPO).

Analysis of carbohydrates and glycoconjugates by matrix‐assisted laser desorption/ionization mass spectrometry: An update for 2019–2020

“…A comparison of three methods for characterizing Nlinked glycosites in the secretome of CHO cells has been reported by Mertz et al (2020). The three methods were (a) the recently developed N-linked glycans and glycosite-containing peptides (NGAG) chemoenzymatic method, (b) solid-phase extraction of N-linked glycoproteins (SPEG), and (c) hydrophilic interaction liquid chromatography (HILIC).…”

Strategies for Glycoengineering Therapeutic Proteins

“…Our team discovered that tri-butanoylated hexosamines, exemplified by 1,3,4-O-Bu 3 ManNAc ( Figure 6D ), provide even higher flux into biosynthetic pathways, increasing sialylation with high efficiency ( Aich et al, 2008 ; Almaraz et al, 2012 ). This analog increases the sialylation of therapeutic proteins including IgG antibodies ( Yin et al, 2017 ), EPO ( Mertz et al, 2020 ) and ENPP-1 ( Stabach et al, 2021 ). In the case of ENPP1-Fc, production with 1,3,4-O-Bu 3 ManNAc increased serum half-life from 170 to 204 h and the AUC from 37,000 to 45,000 ( Figures 3E,F ).…”

“…Today, advances in nucleic acid gene-editing techniques including zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats with Cas9 protein (CRISPR/Cas9) facilitate precise, stable, and systematic engineering of the glycosylation capabilities of mammalian cells ( Narimatsu et al, 2021 ; Wang et al, 2019 ). One example is the over-expression of α2,6-sialyltransferase (ST6) in non-human cell lines such as CHO cells ( Yin et al, 2015 ), which have been used to produce EPO and IgG antibodies ( Mertz et al, 2020 ; Yin et al, 2015 ) as well as ENPP1-Fc with improved sialylation and PK properties [ Figure 3D , ( Stabach et al, 2021 )]. ST6 over-expression increases overall sialylation and results in a humanized α2,6-sialylation profile in CHO cells.…”