TY - JOUR
T1 - One-Step Enrichment of Intact Glycopeptides From Glycoengineered Chinese Hamster Ovary Cells
AU - Yang, Ganglong
AU - Höti, Naseruddin
AU - Chen, Shao Yung
AU - Zhou, Yangying
AU - Wang, Qiong
AU - Betenbaugh, Michael
AU - Zhang, Hui
N1 - Funding Information:
Funding. This work was supported by the National Cancer Institute, the Clinical Proteomic Tumor Analysis Consortium (CPTAC, Grant U24CA210985), the Early Detection Research Network (EDRN, U01CA152813), and the National Science Foundation (Grant 1512265).
Publisher Copyright:
© Copyright © 2020 Yang, Höti, Chen, Zhou, Wang, Betenbaugh and Zhang.
PY - 2020/4/17
Y1 - 2020/4/17
N2 - 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.
AB - 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.
KW - FUT8 knockout
KW - glycoengineered CHO cells
KW - intact glycopeptides
KW - mass spectrometry
KW - one-step enrichment
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U2 - 10.3389/fchem.2020.00240
DO - 10.3389/fchem.2020.00240
M3 - Article
C2 - 32363175
AN - SCOPUS:85084080953
VL - 8
JO - Frontiers in Chemistry
JF - Frontiers in Chemistry
SN - 2296-2646
M1 - 240
ER -