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QUANTITY: An Isobaric Tag for Quantitative Glycomics.

Yang S, Wang M, Chen L, Yin B, Song G, Turko IV, Phinney KW, Betenbaugh MJ, Zhang H, Li S - Sci Rep (2015)

Bottom Line: Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity.Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples.This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

ABSTRACT
Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering.

No MeSH data available.


Regulation of sialic acid in CHO by ST6Gal1(+) and ST3Gal4(−).Same amount of proteins are used for glycan extraction and labeling. (a) Coomassie blue of CHO cell proteins on WT, ST6Gal1(+), and ST3Gal4(−); Lectin blot on WT and ST6Gal1 indicates increased sialic acid expression in ST6Gal1 knock-in CHO cells. (b) Heatmap of sialylated N-glycans from CHO cell glycoproteins on WT, ST6Gal1(+), and ST3Gal4(−). Quantitation is obtained from MS2 of QUANTITY-labeled N-glycans. Increase of sialic acid expression is observed in ST6Gal1(+) while down-regulated expression is observed in ST3Gal4(−), such as (c) N2H2S2, (d) FN2H2S2.
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f5: Regulation of sialic acid in CHO by ST6Gal1(+) and ST3Gal4(−).Same amount of proteins are used for glycan extraction and labeling. (a) Coomassie blue of CHO cell proteins on WT, ST6Gal1(+), and ST3Gal4(−); Lectin blot on WT and ST6Gal1 indicates increased sialic acid expression in ST6Gal1 knock-in CHO cells. (b) Heatmap of sialylated N-glycans from CHO cell glycoproteins on WT, ST6Gal1(+), and ST3Gal4(−). Quantitation is obtained from MS2 of QUANTITY-labeled N-glycans. Increase of sialic acid expression is observed in ST6Gal1(+) while down-regulated expression is observed in ST3Gal4(−), such as (c) N2H2S2, (d) FN2H2S2.

Mentions: A complete list of N-glycans from CHO (WT, ST6Gal1(+), and ST3Gal4(−)) is provided (Fig. 5, Table S4 and Figure S8 & S9). Lectin blot confirmed the up-regulation of sialic acids in ST6Gal1 (+) and the partial down-regulation of sialic acids in ST3Gal4 (−) (Fig. 5a). A total of 159 N-glycans were quantitatively analyzed, in which 114 (71.2%) of the N-glycans were up-regulated in ST6Gal1(+). These over-expressed N-glycans had a greater number of terminal sialic acids, as depicted in the heatmap (Fig. 5b). The most abundant sialylated glycans, including N2H2S, N2H2S2, FN2H2S2, are over-expressed in ST6Gal1(+) while down-regulated in ST3Gal4(−) as expected (Fig. 5c–e). In addition, 44 glycans were only detected in ST6Gal1(+) compared to WT, such as FN5H5S6. In contrast, only 22 (13.8%) glycans were down-regulated in ST3Gal4(−). Three of them (FN2H2S3, FH3, and FN3HS) were completely absent in ST3Gal4(−), while the other 19 glycans (e.g. N3H3S, N2H2S2, FN2H2S2, and FN6H2) were only down-regulated, probably because other enzymes partially compensate the lost ST3Gal4 activity. These results indicate that protein glycosylation in CHO cells can be significantly modified by regulating the expression of a large family of genes that are involved in glycan biosynthesis. This can not only provide a powerful means to quantitatively investigate the in vivo functions of these enzymes, but also lead to engineered CHO cells for the production of therapeutic proteins with desirable pharmaceutical properties.


QUANTITY: An Isobaric Tag for Quantitative Glycomics.

Yang S, Wang M, Chen L, Yin B, Song G, Turko IV, Phinney KW, Betenbaugh MJ, Zhang H, Li S - Sci Rep (2015)

Regulation of sialic acid in CHO by ST6Gal1(+) and ST3Gal4(−).Same amount of proteins are used for glycan extraction and labeling. (a) Coomassie blue of CHO cell proteins on WT, ST6Gal1(+), and ST3Gal4(−); Lectin blot on WT and ST6Gal1 indicates increased sialic acid expression in ST6Gal1 knock-in CHO cells. (b) Heatmap of sialylated N-glycans from CHO cell glycoproteins on WT, ST6Gal1(+), and ST3Gal4(−). Quantitation is obtained from MS2 of QUANTITY-labeled N-glycans. Increase of sialic acid expression is observed in ST6Gal1(+) while down-regulated expression is observed in ST3Gal4(−), such as (c) N2H2S2, (d) FN2H2S2.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4663469&req=5

f5: Regulation of sialic acid in CHO by ST6Gal1(+) and ST3Gal4(−).Same amount of proteins are used for glycan extraction and labeling. (a) Coomassie blue of CHO cell proteins on WT, ST6Gal1(+), and ST3Gal4(−); Lectin blot on WT and ST6Gal1 indicates increased sialic acid expression in ST6Gal1 knock-in CHO cells. (b) Heatmap of sialylated N-glycans from CHO cell glycoproteins on WT, ST6Gal1(+), and ST3Gal4(−). Quantitation is obtained from MS2 of QUANTITY-labeled N-glycans. Increase of sialic acid expression is observed in ST6Gal1(+) while down-regulated expression is observed in ST3Gal4(−), such as (c) N2H2S2, (d) FN2H2S2.
Mentions: A complete list of N-glycans from CHO (WT, ST6Gal1(+), and ST3Gal4(−)) is provided (Fig. 5, Table S4 and Figure S8 & S9). Lectin blot confirmed the up-regulation of sialic acids in ST6Gal1 (+) and the partial down-regulation of sialic acids in ST3Gal4 (−) (Fig. 5a). A total of 159 N-glycans were quantitatively analyzed, in which 114 (71.2%) of the N-glycans were up-regulated in ST6Gal1(+). These over-expressed N-glycans had a greater number of terminal sialic acids, as depicted in the heatmap (Fig. 5b). The most abundant sialylated glycans, including N2H2S, N2H2S2, FN2H2S2, are over-expressed in ST6Gal1(+) while down-regulated in ST3Gal4(−) as expected (Fig. 5c–e). In addition, 44 glycans were only detected in ST6Gal1(+) compared to WT, such as FN5H5S6. In contrast, only 22 (13.8%) glycans were down-regulated in ST3Gal4(−). Three of them (FN2H2S3, FH3, and FN3HS) were completely absent in ST3Gal4(−), while the other 19 glycans (e.g. N3H3S, N2H2S2, FN2H2S2, and FN6H2) were only down-regulated, probably because other enzymes partially compensate the lost ST3Gal4 activity. These results indicate that protein glycosylation in CHO cells can be significantly modified by regulating the expression of a large family of genes that are involved in glycan biosynthesis. This can not only provide a powerful means to quantitatively investigate the in vivo functions of these enzymes, but also lead to engineered CHO cells for the production of therapeutic proteins with desirable pharmaceutical properties.

Bottom Line: Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity.Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples.This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation.

View Article: PubMed Central - PubMed

Affiliation: Department of Pathology, Johns Hopkins University, Baltimore, MD, USA.

ABSTRACT
Glycan is an important class of macromolecules that play numerous biological functions. Quantitative glycomics--analysis of glycans at global level--however, is far behind genomics and proteomics owing to technical challenges associated with their chemical properties and structural complexity. As a result, technologies that can facilitate global glycan analysis are highly sought after. Here, we present QUANTITY (Quaternary Amine Containing Isobaric Tag for Glycan), a quantitative approach that can not only enhance detection of glycans by mass spectrometry, but also allow high-throughput glycomic analysis from multiple biological samples. This robust tool enabled us to accomplish glycomic survey of bioengineered Chinese Hamster Ovary (CHO) cells with knock-in/out enzymes involved in protein glycosylation. Our results demonstrated QUANTITY is an invaluable technique for glycan analysis and bioengineering.

No MeSH data available.