Limits...
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.


Related in: MedlinePlus

Efficiency and linear range of glycan labeling with QUANTITY.N-Glycans extracted from bovine fetuin are prepared in a ratio of 1:1:3:5 prior to labeling with QUANTITY via reduction amination. The labeled fetuin glycans (m/z at 176, 177, 178, and 179), sialylated glycans, including N2H2S2, N3H3S2, N3H3S3, and N3H3S4, are pooled for electrospray (ESI) – tandem mass spectrometry (MS2) (note: core structure N2H3 is not included in the composition). (a) MS spectrum of fetuin N-glycans. (b) MS2 spectra of N2H2S2 consist of glycan fragmentation and QUANTITY reporter ions. (c) Linear range of glycan-QUANTITY labeling at a ratio of 1:1:3:5. Error bar were obtained from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4663469&req=5

f3: Efficiency and linear range of glycan labeling with QUANTITY.N-Glycans extracted from bovine fetuin are prepared in a ratio of 1:1:3:5 prior to labeling with QUANTITY via reduction amination. The labeled fetuin glycans (m/z at 176, 177, 178, and 179), sialylated glycans, including N2H2S2, N3H3S2, N3H3S3, and N3H3S4, are pooled for electrospray (ESI) – tandem mass spectrometry (MS2) (note: core structure N2H3 is not included in the composition). (a) MS spectrum of fetuin N-glycans. (b) MS2 spectra of N2H2S2 consist of glycan fragmentation and QUANTITY reporter ions. (c) Linear range of glycan-QUANTITY labeling at a ratio of 1:1:3:5. Error bar were obtained from three independent experiments.

Mentions: Next, we tested the quantification accuracy of QUANTITY by labeling fetuin N-glycans with 4-plex QUANTITY at a ratio of 1:1:3:5. Fetuin proteins from fetal bovine (200 μg, 200 μg, 600 μg, and 1000 μg; triplicates) were processed by following our standard protocol. Figure 3a shows the MS1 of QUANTITY labeled fetuin N-glycans, including N2H2S2, N3H3S2, N3H3S3, and N3H3S4. Figure 3b is a representative full MS2 spectrum (N2H2S2), which includes a series of glycan fragments for easy structural elucidation of the precursor ion and strong reporter ions ranging from 176 to 179. The inset is the expanded low mass range of the MS2 showing the signal of each reporter ion, which indicates the relative abundance of glycans from four original samples. The experimental result of this glycan (N2H2S2) is very close to 1:1:3:5 (Fig. 3c). The linear correlation between the measured and theoretical ratios and the small standard deviation from three independent replicates indicate the great reproducibility of QUANTITY quantification (the quantification of other glycans is provided in the supporting information Table S1).


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)

Efficiency and linear range of glycan labeling with QUANTITY.N-Glycans extracted from bovine fetuin are prepared in a ratio of 1:1:3:5 prior to labeling with QUANTITY via reduction amination. The labeled fetuin glycans (m/z at 176, 177, 178, and 179), sialylated glycans, including N2H2S2, N3H3S2, N3H3S3, and N3H3S4, are pooled for electrospray (ESI) – tandem mass spectrometry (MS2) (note: core structure N2H3 is not included in the composition). (a) MS spectrum of fetuin N-glycans. (b) MS2 spectra of N2H2S2 consist of glycan fragmentation and QUANTITY reporter ions. (c) Linear range of glycan-QUANTITY labeling at a ratio of 1:1:3:5. Error bar were obtained from three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

f3: Efficiency and linear range of glycan labeling with QUANTITY.N-Glycans extracted from bovine fetuin are prepared in a ratio of 1:1:3:5 prior to labeling with QUANTITY via reduction amination. The labeled fetuin glycans (m/z at 176, 177, 178, and 179), sialylated glycans, including N2H2S2, N3H3S2, N3H3S3, and N3H3S4, are pooled for electrospray (ESI) – tandem mass spectrometry (MS2) (note: core structure N2H3 is not included in the composition). (a) MS spectrum of fetuin N-glycans. (b) MS2 spectra of N2H2S2 consist of glycan fragmentation and QUANTITY reporter ions. (c) Linear range of glycan-QUANTITY labeling at a ratio of 1:1:3:5. Error bar were obtained from three independent experiments.
Mentions: Next, we tested the quantification accuracy of QUANTITY by labeling fetuin N-glycans with 4-plex QUANTITY at a ratio of 1:1:3:5. Fetuin proteins from fetal bovine (200 μg, 200 μg, 600 μg, and 1000 μg; triplicates) were processed by following our standard protocol. Figure 3a shows the MS1 of QUANTITY labeled fetuin N-glycans, including N2H2S2, N3H3S2, N3H3S3, and N3H3S4. Figure 3b is a representative full MS2 spectrum (N2H2S2), which includes a series of glycan fragments for easy structural elucidation of the precursor ion and strong reporter ions ranging from 176 to 179. The inset is the expanded low mass range of the MS2 showing the signal of each reporter ion, which indicates the relative abundance of glycans from four original samples. The experimental result of this glycan (N2H2S2) is very close to 1:1:3:5 (Fig. 3c). The linear correlation between the measured and theoretical ratios and the small standard deviation from three independent replicates indicate the great reproducibility of QUANTITY quantification (the quantification of other glycans is provided in the supporting information Table S1).

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.


Related in: MedlinePlus