One-Step Enrichment of Intact Glycopeptides From Glycoengineered Chinese Hamster Ovary Cells

Overview

Journal Front Chem

Specialty Chemistry

Date 2020 May 5

PMID 32363175

Citations 10

Authors

Ganglong Yang

Naseruddin Hoti

Shao-Yung Chen

Yangying Zhou

Qiong Wang

Michael Betenbaugh

Hui Zhang

Affiliations

Soon will be listed here.

Abstract

Recently, the glycoproteomic analysis of intact glycopeptides has emerged as an effective approach to decipher the glycan modifications of glycoproteins at the site-specific level. A rapid method to enrich intact glycopeptides is essential for the analysis of glycoproteins, especially for biopharmaceutical proteins. In this study, we established a one-step method for the rapid capture of intact glycopeptides for analysis by mass spectrometry. Compared to the conventional sequential enrichment method, the one-step intact glycopeptide enrichment method reduced the sample preparation time and improved the detection of intact glycopeptides with long sequences or non-polar amino acids. Moreover, an increased number of glycosite-containing peptides was identified by the one-step method compared with the sequential method. When we applied this method to the glycoproteomic analysis of glycoengineered Chinese hamster ovary (CHO)-K1 cells with α1,6-fucosyltransferase () knockout, the results showed that the knockout of altered the overall glycosylation profile of CHO-K1 cells with the elimination of core fucosylation and together with increases in high-mannose and sialylated N-glycans. Interestingly, the knockout of the also appeared to regulate the expression of glycoproteins involved in several functions and pathways in CHO-K1 cells, such as the down-regulation of an intracellular lectin LMAN2 showing cellular adaptation to the alterations in knockout cells. These findings indicate that the site-specific characterization of glycoproteins from glycoengineered CHO-K1 cells can be achieved rapidly using the one-step intact glycopeptide enrichment method, which could provide insights for bio-analysts and biotechnologists to better tailor therapeutic drugs.

Citing Articles

Characterization of core fucosylation via sequential enzymatic treatments of intact glycopeptides and mass spectrometry analysis.

Cao L, Lih T, Hu Y, Schnaubelt M, Chen S, Zhou Y Nat Commun. 2022; 13(1):3910.

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Strategies for Proteome-Wide Quantification of Glycosylation Macro- and Micro-Heterogeneity.

Fang P, Ji Y, Oellerich T, Urlaub H, Pan K Int J Mol Sci. 2022; 23(3).

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Methods for quantification of glycopeptides by liquid separation and mass spectrometry.

Yin H, Zhu J Mass Spectrom Rev. 2022; 42(2):887-917.

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Glycoproteomic Characterization of FUT8 Knock-Out CHO Cells Reveals Roles of FUT8 in the Glycosylation.

Yang G, Wang Q, Chen L, Betenbaugh M, Zhang H Front Chem. 2021; 9:755238.

PMID: 34778211 PMC: 8586412. DOI: 10.3389/fchem.2021.755238.

ZIC-cHILIC Functionalized Magnetic Nanoparticle for Rapid and Sensitive Glycopeptide Enrichment from <1 µL Serum.

Pradita T, Chen Y, Mernie E, Bendulo S, Chen Y Nanomaterials (Basel). 2021; 11(9).

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