Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells

Yang, Ganglong; Hu, Yingwei; Sun, Shisheng; OuYang, Chuanzi; Yang, Weiming; Wang, Qiong; Betenbaugh, Michael J.; Zhang, Hui

doi:10.1021/acs.analchem.8b03520

Cited by 43 publications

(38 citation statements)

References 62 publications

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“…The mass spectrometric method and data analysis are described in our previous publication with modification (Yang et al, 2018). In brief, the peptide samples in 3% ACN and 0.1% FA were analyzed with a Q-Exactive or Orbitrap Fusion Lumos Tribrid mass spectrometer (Thermo Fisher Scientific).…”

Section: Nano-lc-ms/ms Analysis and Data Analysismentioning

confidence: 99%

“…Then, the IGPs were eluted with 50% ACN from the one-step method. Meanwhile, these two one-step enrichment strategies were also compared with a traditional sequential method using two individual enrichment steps (sequential method) that we described in our previous studies (Yang et al, 2017(Yang et al, , 2018, in which the peptides were prepared by hydrophobic chemistry using C18 column, eluted with 60% ACN. The eluted peptides were dried, and the IGPs were enriched by hydrophilic chemistry using a MAX column.…”

Section: Rapid Capture Of Intact Glycopeptides From Fetuin By the Onementioning

confidence: 99%

“…Briefly, the bovine fetuin was first digested wit trypsin, and the tryptic peptides were enriched for IGPs by the one-step methods (mixed or stacked columns) and the traditional sequential glycopeptide enrichment method (sequential method), respectively. The peptides with and without glycopeptide enrichment were run on a Q-Exactive mass spectrometer using our previous IGP analysis parameters (Yang et al, 2018). The glycosite-containing database was comprised of three N-linked glycosite-containing peptides from bovine fetuin (P12763, up to two missed cleavage sites) ( Table S1).…”

Section: Rapid Capture Of Intact Glycopeptides From Fetuin By the Onementioning

confidence: 99%

“…The enriched glycopeptides were analyzed twice using LC-MS/MS. We previously constructed a sample-specific glycopeptide database for the identification of N-linked glycosite-containing peptides using PNGase-F-released peptides (Yang et al, 2018). We applied the previous CHO-K1 cell glycomic results as glycan database (North et al, 2010).…”

Section: Analysis Of Intact Glycopeptides From the Wild-type And Fut8mentioning

confidence: 99%

“…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). Recently, the mass spectrometric analysis of intact glycopeptides has emerged as a promising strategy to analyze glycoproteins for glycan modifications at site-and structure-specific levels (Sun et al, 2016;Yang et al, 2018;Xiao and Tian, 2019). However, the major limitation for this approach is the low detectability of intact glycopeptides in a complex mixture, which are suppressed by non-glycosylated peptides (Shajahan et al, 2017).…”

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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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.

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Section: Nano-lc-ms/ms Analysis and Data Analysismentioning

confidence: 99%

Section: Rapid Capture Of Intact Glycopeptides From Fetuin By the Onementioning

confidence: 99%

Section: Rapid Capture Of Intact Glycopeptides From Fetuin By the Onementioning

confidence: 99%

Section: Analysis Of Intact Glycopeptides From the Wild-type And Fut8mentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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

et al. 2020

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Characterization of the Ubiquitin‐Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress

Selvaprakash,

Sideri,

Henry

et al. 2024

Biotechnology Journal

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Chinese hamster ovary (CHO) cells remain the most widely used host cell line for biotherapeutics production. Despite their widespread use, understanding endoplasmic reticulum (ER) stress conditions in recombinant protein production remains limited, often creating bottlenecks preventing improved production titers and product quality. Ubiquitination not only targets substrates (e.g., misfolded proteins) for proteasome degradation but also has important regulatory control functions including cell cycle regulation, translation, apoptosis, autophagy, etc. and hence is likely to be central to understanding and controlling the productivity of recombinant biotherapeutics. This study aimed to uncover differentially expressed ubiquitinated proteins following artificial induction of ER‐stress in recombinant CHO cells. CHO cells were treated with the stress inducer tunicamycin and the proteasome inhibitor MG132, followed by LC‐MS/MS proteomic analysis. We identified >4000 ubiquitinated peptides from CHO‐DP12 cells under ER stress conditions and proteasome inhibition. Moreover, data analysis showed altered abundance levels of >900 ubiquitinated proteins under the combination of ER stress and proteasome inhibition compared to untreated controls. Gene Ontology (GO) analysis of these ubiquitinated proteins resulted in a significant enrichment of key pathways involving the proteasome, protein processing in the ER, N‐glycan biosynthesis, and ubiquitin‐mediated proteolysis. ER stress response proteins such as GRP78, HSP90B1, ATF6, HERPUD1, and PDIA4 were found to be highly ubiquitinated and exhibited a significant increase in abundance following induction of ER‐stress conditions. This study broadens our comprehension of the roles played by protein ubiquitination in CHO cell stress responses, potentially revealing targets for tailored cell line engineering aimed at enhancing stress tolerance and production efficiency.

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Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

Wang

Yang

Wang

et al. 2019

Biotech & Bioengineering

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With the increasing demand to provide more detailed quality attributes, more sophisticated glycan analysis tools are highly desirable for biopharmaceutical manufacturing. Here, we performed an intact glycopeptide analysis method to simultaneously analyze the site-specific N-and O-glycan profiles of the recombinant erythropoietin Fc (EPO-Fc) protein secreted from a Chinese hamster ovary glutamine synthetase stable cell line and compared the effects of two commercial culture media, EX-CELL (EX) and immediate advantage (IA) media, on the glycosylation profile of the target protein. EPO-Fc, containing the Fc region of immunoglobulin G1 (IgG1) fused to EPO, was harvested at Day 5 and 8 of a batch cell culture process followed by purification and N-and O-glycopeptide profiling. A mixed anion exchange chromatographic column was implemented to capture and enrich N-linked glycopeptides. Using intact glycopeptide characterization, the EPO-Fc was observed to maintain their individual EPO and Fc N-glycan characteristics in which the EPO region presented bi-, tri-, and tetra-branched N-glycan structures, while the Fc Nglycan displayed mostly biantennary glycans. EPO-Fc protein generated in EX medium produced more complex tetra-antennary N-glycans at each of the three EPO N-sites while IA medium resulted in a greater fraction of bi-and tri-antennary N-glycans at these same sites. Interestingly, the sialylation content decreased from sites 1-4 in both media while the fucosylation progressively increased with a maximum at the final IgG Fc site. Moreover, we observed that low amounts of Neu5Gc were detected and the content increased at the later sampling time in both EX and IA media. For O-glycopeptides, both media produced predominantly three structures, N1F1F0SOG0, N1H1F0S1G0, and N1H1F0S2G0, with lesser amounts of other structures. This intact glycopeptide method can decipher site-specific glycosylation profile and provide a more detailed characterization of N-and O-glycans present for enhanced understanding of the key product quality attributes such as media on recombinant proteins of biotechnology interest. K E Y W O R D S CHO-GS cells, culture media, EPO-Fc protein, intact glycopeptide analysis, site-specific glycosylation SUPPORTING INFORMATION Additional supporting information may be found online in the Supporting Information section.

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Comprehensive Glycoproteomic Analysis of Chinese Hamster Ovary Cells

Cited by 43 publications

References 62 publications

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

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

Characterization of the Ubiquitin‐Modified Proteome of Recombinant Chinese Hamster Ovary Cells in Response to Endoplasmic Reticulum Stress

Characterization of intact glycopeptides reveals the impact of culture media on site‐specific glycosylation of EPO‐Fc fusion protein generated by CHO‐GS cells

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