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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.


MS profiling and MS/MS quantitation of QUANTITY-labeled glycans from ST6Gal1(+) and ST3Gal4(−) CHO cells.N-Glycans are extracted using method described in Fig. 1. (a) MS1 spectrum of CHO glycans with multiple charges. (b) MS1 spectrum of CHO glycans after converting to single charge (Xcalibur). (c) MS2 spectrum of one sialylated glycan, N2H2S. (d) Quantitation using reporter ions from four QUANTITY-labeled glycans.
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f4: MS profiling and MS/MS quantitation of QUANTITY-labeled glycans from ST6Gal1(+) and ST3Gal4(−) CHO cells.N-Glycans are extracted using method described in Fig. 1. (a) MS1 spectrum of CHO glycans with multiple charges. (b) MS1 spectrum of CHO glycans after converting to single charge (Xcalibur). (c) MS2 spectrum of one sialylated glycan, N2H2S. (d) Quantitation using reporter ions from four QUANTITY-labeled glycans.

Mentions: We cultured three cell lines in F12-K medium with fetal bovine serum, except for ST6Gal1(+) in which blasticidin was also included. Glycans extracted from these cells were analyzed by using our standard solid phase based protocol (Figure S8)23. Most QUANTITY-labeled glycans exhibited multiple charges (+2 and +3) and gave satisfactory MS2 fragments for structural determination (Fig. 4a,c). After all precursor ions were extracted as singly-charged peaks by Thermo Xcalibur-Xtract (Fig. 4b), each glycan only consisted of a single species and no metal adducts were observed. This feature, unique to QUANTITY-labeled glycans, was significant in that glycans could otherwise form multiple metal adducts that average out their intensity to more species and reduce their detection sensitivity. Figure 4c is the representative full MS2 spectrum of N2H2S showing strong signal on reporter ions for its quantification and distinct large fragments for its structural identification. The reporter ions of several other glycans, including FN2H2S2 (2782.2), N2H2S2 (2635.1), N3H3S3 (3380.4), and N5H4S4 (4329.0), are also shown in Fig. 4d.


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)

MS profiling and MS/MS quantitation of QUANTITY-labeled glycans from ST6Gal1(+) and ST3Gal4(−) CHO cells.N-Glycans are extracted using method described in Fig. 1. (a) MS1 spectrum of CHO glycans with multiple charges. (b) MS1 spectrum of CHO glycans after converting to single charge (Xcalibur). (c) MS2 spectrum of one sialylated glycan, N2H2S. (d) Quantitation using reporter ions from four QUANTITY-labeled glycans.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f4: MS profiling and MS/MS quantitation of QUANTITY-labeled glycans from ST6Gal1(+) and ST3Gal4(−) CHO cells.N-Glycans are extracted using method described in Fig. 1. (a) MS1 spectrum of CHO glycans with multiple charges. (b) MS1 spectrum of CHO glycans after converting to single charge (Xcalibur). (c) MS2 spectrum of one sialylated glycan, N2H2S. (d) Quantitation using reporter ions from four QUANTITY-labeled glycans.
Mentions: We cultured three cell lines in F12-K medium with fetal bovine serum, except for ST6Gal1(+) in which blasticidin was also included. Glycans extracted from these cells were analyzed by using our standard solid phase based protocol (Figure S8)23. Most QUANTITY-labeled glycans exhibited multiple charges (+2 and +3) and gave satisfactory MS2 fragments for structural determination (Fig. 4a,c). After all precursor ions were extracted as singly-charged peaks by Thermo Xcalibur-Xtract (Fig. 4b), each glycan only consisted of a single species and no metal adducts were observed. This feature, unique to QUANTITY-labeled glycans, was significant in that glycans could otherwise form multiple metal adducts that average out their intensity to more species and reduce their detection sensitivity. Figure 4c is the representative full MS2 spectrum of N2H2S showing strong signal on reporter ions for its quantification and distinct large fragments for its structural identification. The reporter ions of several other glycans, including FN2H2S2 (2782.2), N2H2S2 (2635.1), N3H3S3 (3380.4), and N5H4S4 (4329.0), are also shown in Fig. 4d.

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.