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

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.

High‐Throughput Analyses of Glycans, Glycosites, and Intact Glycopeptides Using C4‐and C18/MAX‐Tips and Liquid Handling System

“…We have reported that for a column‐based elution, the related position of C18 and MAX resin are optimized if they are stacked with C18 on top (G. Yang et al., 2020 ). However, because liquid handling systems would aspirate and disperse in an up‐down movement (unlike column‐based flow, which generally uses gravity to allow the elution wash to flow naturally).…”

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

“…Mass spectrometry (MS) has been used as a prime tool for full characterization of the glycan structures, glycosylation site, and protein carrier on glycoprotein. However, due to low abundance, heterogeneity, and low detectability caused by the ionization suppression from non-glycosylated peptides, direct analysis of glycopeptides is still challenging [3,11,12]. Various glycopeptide enrichment strategies prior to MS analysis such as lectin affinity chromatography [13][14][15], hydrazide chemistry [16][17][18], boronic acid [19][20][21][22], and hydrophilic interaction chromatography [23][24][25][26] have been widely studied.…”

An Ultrafast N-Glycoproteome Analysis Method Using Thermoresponsive Magnetic Fluid-Immobilized Enzymes

“…Glycosylation is one of the most prominent post-translational modification methods for proteins (Stadlmann et al, 2017 ; Huang et al, 2019 ). As a major type, N- glycosylation has a wide range of functions that greatly amplifies the diversity of proteins (Hart and Copeland, 2010 ; Yang et al, 2020 ). From the general biological process, such as cell adhesion and signal transduction, to specific functions of proteins like folding and stability, the complexity imparted to a proteome by N- glycosylation is immense (Schjoldager et al, 2020 ).…”