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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.


Comparison of N-glycan labeling by QUANTITY and aminoxyTMT-126 (TMT).Standard sialylglycopeptide (SGP) was used for labeling via reduction amination (1M NaCHBH3, DMSO:HOAc = 7:3, 65 °C/4h). SGP has two sialic acids including N2H2S and N2H2S2. (a) Full MS spectrum of SGP-QUANTITY. (b) MS1 spectrum of N2H2S2-QUANTITY. (c) MS2 reporter ions of N2H2S2-QUANTITY. (d) Full MS spectrum of SGP-TMT. (e) MS1 spectrum of N2H2S2-TMT, several by-products observed after aminoxyTMT labeling. (f) MS2 reporter ions of N2H2S2-TMT.
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f2: Comparison of N-glycan labeling by QUANTITY and aminoxyTMT-126 (TMT).Standard sialylglycopeptide (SGP) was used for labeling via reduction amination (1M NaCHBH3, DMSO:HOAc = 7:3, 65 °C/4h). SGP has two sialic acids including N2H2S and N2H2S2. (a) Full MS spectrum of SGP-QUANTITY. (b) MS1 spectrum of N2H2S2-QUANTITY. (c) MS2 reporter ions of N2H2S2-QUANTITY. (d) Full MS spectrum of SGP-TMT. (e) MS1 spectrum of N2H2S2-TMT, several by-products observed after aminoxyTMT labeling. (f) MS2 reporter ions of N2H2S2-TMT.

Mentions: We first demonstrated the completion of QUANTITY labeling. Two N-glycans (N2H2S2 > 90%, and N2H2S < 10%) extracted from 1 mg standard sialylglycopeptide (SGP) were labeled with QUANTITY-176 (30 μL at 40 mM) in 70% dimethyl sulfoxide (DMSO) and 30% acetic acid (HOAc) containing 1 M sodium cyanoborohydride (NaCNBH3). As a comparison, the SGP glycans were also labeled by a commercially available isobaric tag for glycans based on tertiary amine (aminoxyTMT-126) similarly. Figure 2 shows the MS1 (a & b) and MS2 (c) of QUANTITY-labeled SGP glycans, as well as the MS1 (d & e) and MS2 (f) of aminoxyTMT labeled counterparts acquired on an ESI instrument (their MALDI MS1 spectra, together with the MALDI MS1 of unlabeled SGP glycans, are also available in Figure S4). The QUANTITY-labeled glycans only show two dominant peaks as labeled N2H2S and N2H2S2, indicating the labeling reaction was completed. Although aminoxyTMT-labeled glycans also show two major peaks, we still observed unlabeled N2H2S and N2H2S2, suggesting the labeling reaction with aminoxyTMT was more difficult to complete. In addition, the aminoxyTMT labeled glycans display multiple satellite peaks with 14 Dalton mass difference (Fig. 2e), making MS1 more complex and averaging out peak intensity. More significantly, when the labeled glycans were fragmented, the reporter ion (m/z 176.11) generated from the quaternary amine of QUANTITY yields decent signal in comparison to common glycan fragment ions (m/z 138.05 or 204.09). In contrast, the reporter ion (m/z 126.11) generated from the tertiary amine of aminoxyTMT not only shows much less intensity, but is susceptible to the interference from a glycan fragment (m/z 126.05) (Fig. 2c,f). This experiment clearly demonstrated the completion of QUANTITY labeling and its advantages over the existing isobaric tags for glycans.


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)

Comparison of N-glycan labeling by QUANTITY and aminoxyTMT-126 (TMT).Standard sialylglycopeptide (SGP) was used for labeling via reduction amination (1M NaCHBH3, DMSO:HOAc = 7:3, 65 °C/4h). SGP has two sialic acids including N2H2S and N2H2S2. (a) Full MS spectrum of SGP-QUANTITY. (b) MS1 spectrum of N2H2S2-QUANTITY. (c) MS2 reporter ions of N2H2S2-QUANTITY. (d) Full MS spectrum of SGP-TMT. (e) MS1 spectrum of N2H2S2-TMT, several by-products observed after aminoxyTMT labeling. (f) MS2 reporter ions of N2H2S2-TMT.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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f2: Comparison of N-glycan labeling by QUANTITY and aminoxyTMT-126 (TMT).Standard sialylglycopeptide (SGP) was used for labeling via reduction amination (1M NaCHBH3, DMSO:HOAc = 7:3, 65 °C/4h). SGP has two sialic acids including N2H2S and N2H2S2. (a) Full MS spectrum of SGP-QUANTITY. (b) MS1 spectrum of N2H2S2-QUANTITY. (c) MS2 reporter ions of N2H2S2-QUANTITY. (d) Full MS spectrum of SGP-TMT. (e) MS1 spectrum of N2H2S2-TMT, several by-products observed after aminoxyTMT labeling. (f) MS2 reporter ions of N2H2S2-TMT.
Mentions: We first demonstrated the completion of QUANTITY labeling. Two N-glycans (N2H2S2 > 90%, and N2H2S < 10%) extracted from 1 mg standard sialylglycopeptide (SGP) were labeled with QUANTITY-176 (30 μL at 40 mM) in 70% dimethyl sulfoxide (DMSO) and 30% acetic acid (HOAc) containing 1 M sodium cyanoborohydride (NaCNBH3). As a comparison, the SGP glycans were also labeled by a commercially available isobaric tag for glycans based on tertiary amine (aminoxyTMT-126) similarly. Figure 2 shows the MS1 (a & b) and MS2 (c) of QUANTITY-labeled SGP glycans, as well as the MS1 (d & e) and MS2 (f) of aminoxyTMT labeled counterparts acquired on an ESI instrument (their MALDI MS1 spectra, together with the MALDI MS1 of unlabeled SGP glycans, are also available in Figure S4). The QUANTITY-labeled glycans only show two dominant peaks as labeled N2H2S and N2H2S2, indicating the labeling reaction was completed. Although aminoxyTMT-labeled glycans also show two major peaks, we still observed unlabeled N2H2S and N2H2S2, suggesting the labeling reaction with aminoxyTMT was more difficult to complete. In addition, the aminoxyTMT labeled glycans display multiple satellite peaks with 14 Dalton mass difference (Fig. 2e), making MS1 more complex and averaging out peak intensity. More significantly, when the labeled glycans were fragmented, the reporter ion (m/z 176.11) generated from the quaternary amine of QUANTITY yields decent signal in comparison to common glycan fragment ions (m/z 138.05 or 204.09). In contrast, the reporter ion (m/z 126.11) generated from the tertiary amine of aminoxyTMT not only shows much less intensity, but is susceptible to the interference from a glycan fragment (m/z 126.05) (Fig. 2c,f). This experiment clearly demonstrated the completion of QUANTITY labeling and its advantages over the existing isobaric tags for glycans.

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.