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

Ganglong Yang,Shao-Yung Chen,Yangying Zhou,Naseruddin Höti,Michael Betenbaugh,Hui Zhang,Qiong Wang

doi:10.3389/fchem.2020.00240

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 (FUT8) knockout, the results showed that the knockout of FUT8 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 FUT8 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 FUT8 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.

Highlights

It has been challenging to interpret the heterogeneity of glycoproteins, which includes protein sequence, glycosite information, glycan structures at each glycosite, and overall and individual glycan occupancy at specific sites (Liu et al, 2017)
Rapid Capture of Intact Glycopeptides From Fetuin by the One-Step Enrichment Method
Using the mixed column or stacked column, the intact glycopeptide (IGP) were enriched from the mixture of peptides by the one-step method, in which the peptides were bound to C18 particles in the column, desalted by washing with 5% ACN, and eluted with 95% ACN

Summary

Introduction

It has been challenging to interpret the heterogeneity of glycoproteins, which includes protein sequence, glycosite information, glycan structures at each glycosite, and overall and individual glycan occupancy at specific sites (Liu et al, 2017). One-Step Enrichment of Intact Glycopeptides modifications at site- and structure-specific levels (Sun et al, 2016; Yang et al, 2018; Xiao and Tian, 2019). A capture method for intact glycopeptides before mass spectrometry analysis is highly desirable. Several approaches, including the immobilization of glycopeptides using hydrazide chemistry (Zhang et al, 2003; Nilsson et al, 2009), lectin enrichment (Kaji et al, 2003; Yang et al, 2013), hydrophilic interaction chromatography (HILIC) (Wada et al, 2004; Sun et al, 2016), and anion exchange (Yang et al, 2017), are reported for glycopeptide capture. The proteins are digested to peptides by protease(s), followed by peptides clean-up by hydrophobic chromatography, drying, and reconstitution in the organic solvent for glycopeptide enrichment using hydrophilic enrichment

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Results

Conclusion