Scientific Reports
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2017
·Vol. 7
·期 1
·1-12
·
IF 4.259
Wu Zhaoguan,Li Henghui,Zhang Qiwei,Liu Xin,Zheng Qi,Li Jianjun
O -Acetylation of sialic acid in protein N-glycans is an important modification and can occur at either 4-, 7-, 8- or 9-position in various combinations. This modification is usually labile under alkaline reaction conditions. Consequently, a permethylation-based analytical method, which has been widely used in glycomics studies, is not suitable for profiling O -acetylation of sialic acids due to the harsh reaction conditions. Alternatively, methylamidation can be used for N-glycan analysis without affecting the base-labile modification of sialic acid. In this report, we applied both permethylation and methylamidation approaches to the analysis of O -acetylation in sialic acids. It has been demonstrated that methylamidation not only stabilizes sialic acids during MALDI processing but also allow for characterization of their O -acetylation pattern. In addition, LC-MS/MS experiments were carried out to distinguish between the O -acetylated glycans with potential isomeric structures. The repeatability of methylamidation was examined to evaluate the applicability of the approach to profiling of O -acetylation in sialic acids. In conclusion, the combination of methylamidation and permethylation methodology is a powerful MALDI-TOF MS-based tool for profiling O -acetylation in sialic acids applicable to screening of N-glycans.
研究领域:
生物化学
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分析化学
·
糖组学
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DOI
PubMed
高影响因子
Analytical Methods
·
2024
·Vol. 16
·期 16
·2472-2477
·
IF 2.7
Chenhao Zhao,Xingdan Wang,Jing Wu,Yeli Hu,Qiwei Zhang,Qi Zheng
Sialic acids are a family of monosaccharides that share a nine-carbon backbone and a carboxyl group. A recent derivatization method based on 3-nitrophenylhydrazine (3-NPH) provides a mild chemical labeling technique for biomolecules containing carbonyl or carboxyl groups. In this study, we utilized 3-NPH to label sialic acids via a two-step derivatization process. The derivatized species can produce a common reporter ion corresponding to C1–C3 with two labels, and a fragment differentiating between Neu5Ac, Neu5Gc, and KDN. This method is compatible with O-acetylated sialic acids and provides high sensitivity to Neu5Gc and KDN, and since the utilization of dual labeling significantly enhances the hydrophobicity of derivatives, it can effectively mitigate matrix effects when combined with parallel reaction monitoring technology. Negative-ion tandem mass spectrometry (MS/MS) analysis reveals a distinctive fragmentation profile for the 4-O-acetylated species, while the other sialic acids yield similar MS/MS spectra with a high abundance of reporter ions. Using the reporter ion as a transition, this analytical strategy is effective for analyzing complex biological samples. For example, it was successfully employed to quantify sialic acids in the intestinal tissues of several carp species, demonstrating its potential in sialylation research.
研究领域:
分析化学
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糖生物学
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PubMed
高影响因子
ACS Omega
·
2025
·
IF 4.3
Zhenghu Min,Xingdan Wang,Xiaoqiu Yang,Qiwei Zhang,Qi Zheng
Glycosylation profiling is an effective methodology for achieving a comprehensive understanding of glycoproteins and their alterations in a multitude of pathological conditions. However, in comparison to N-glycosylation, O-glycosylation presents significant challenges in terms of both qualitative and quantitative mass spectrometric analyses. A recently developed oxidative release protocol enables the selective formation of O-glycans containing a carboxyl group derived from the amino acid residue. In this study, 3-nitrophenylhydrazine was used to derivatize the common carboxyl group in a mild hydrophilic solution. Derivatization resulted in the generation of a series of report ions for serine, threonine, sialic acid, and O-acetylated sialic acid residues, thereby facilitating the identification of O-glycans and their attached amino acid residues, as well as the determination of the number of O-acetyl groups. A total of 65 O-glycans can be identified from bovine mucin. Furthermore, the analytical strategy revealed that O-acetylated N-acetylneuraminic acid (Neu5Ac)-containing O-glycans from horse serum exhibited distinctive fragmentation patterns in comparison to those from bovine mucin. Additionally, the presence of deaminoneuraminic acid (KDN)-containing O-glycans was successfully confirmed in fish intestinal tissue. These findings suggest that this method provides an economical and potentially valuable tool for large-scale O-glycosylation studies in complex biological samples.
研究领域:
生物化学
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蛋白质组学
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分析化学
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PubMed
高影响因子
Analytical Methods
·
2020
·Vol. 12
·期 17
·2221-2227
·
IF 2.596
Junjie Du,Qiwei Zhang,Jianjun Li,Qi Zheng
Sialic acid (Sia) is widespread in vertebrates and humans as well as in some microbes and invertebrates. It is well known that O-acetylation is a common modification for Sia. Also, highly O-acetylated Sias have been reported in humans, bovines, and fish. Derivatization based on 1,2-diamino-4,5-methylenedioxybenzene (DMB) coupled with LC-fluorescence detection or LC-MS analysis has been the routine analytical strategy for O-acetylation profiling of Sias. Unfortunately, DMB offers poor chromatographic resolution for some O-acetylated species. Herein, we report another derivatization reagent, 4,5-dimethylbenzene-1,2-diamine (DMBA), for the LC-MS analysis of Sia isomers. The DMBA method was first evaluated using Sia reference standard, horse serum and carp blood. The results demonstrate that DMBA provides superior chromatographic separation efficiency and comparable MS/MS spectra to DMB. The proposed method was then applied to the analysis of relative abundance of each Sia species in fish tissues, including liver, intestine, muscle and brain. It was found that the relative amounts of Sia species were significantly different among the four tissues. Thus, DMBA derivatization-based LC-MS methods have great potential in investigating the O-acetylation level of Sias in various samples, such as human food and bio-pharmaceutical products.
研究领域:
分析化学
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生物化学
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药学
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高影响因子
JOURNAL OF VIROLOGY
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2023
·Vol. 97
·期 5
·
IF 5.4
Renrong You,Kangchengyin Liu,Min Huang,Lihua Tang,Xuehui Zhang,Yahui Huang,Jing Zhao,Ye Zhao,Lilin Ye,Guozhong Zhang
Infectious bronchitis virus (IBV) infections are initiated by the transmembrane spike (S) glycoprotein, which binds to host factors and fuses the viral and cell membranes. The N-terminal domain of the S1 subunit of IBV S protein binds to sialic acids, but the precise location of the sialic acid binding domain (SABD) and the role of the SABD in IBV-infected chickens remain unclear. Here, we identify the S1 N-terminal amino acid (aa) residues 19 to 227 (209 aa total) of IBV strains SD (GI-19) and GD (GI-7), and the corresponding region of M41 (GI-1), as the minimal SABD using truncated protein histochemistry and neuraminidase assays. Both α-2,3- and α-2,6-linked sialic acids on the surfaces of CEK cells can be used as attachment receptors by IBV, leading to increased infection efficiency. However, 9-O acetylation of the sialic acid glycerol side chain inhibits IBV S1 and SABD protein binding. We further constructed recombinant strains in which the S1 gene or the SABD in the GD and SD genomes were replaced with the corresponding region from M41 by reverse genetics. Infecting chickens with these viruses revealed that the virulence and nephrotropism of rSDM41-S1, rSDM41-206, rGDM41-S1, and rGDM41-206 strains were decreased to various degrees compared to their parental strains. A positive sera cross-neutralization test showed that the serotypes were changed for the recombinant viruses. Our results provide insight into IBV infection of host cells that may aid vaccine design.IMPORTANCE To date, only α-2,3-linked sialic acid has been identified as a potential host binding receptor for IBV. Here, we show the minimum region constituting the sialic acid binding domain (SABD) and the binding characteristics of the S1 subunit of spike (S) protein of IBV strains SD (GI-19), GD (GI-7), and M41 (GI-1) to various sialic acids. The 9-O acetylation modification partially inhibits IBV from binding to sialic acid, while the virus can also bind to sialic acid molecules linked to host cells through an α-2,6 linkage, serving as another receptor determinant. Substitution of the putative SABD from strain M41 into strains SD and GD resulted in reduced virulence, nephrotropism, and a serotype switch. These findings suggest that sialic acid binding has diversified during the evolution of γ-coronaviruses, impacting the biological properties of IBV strains. Our results offer insight into the mechanisms by which IBV invades host cells.
研究领域:
病毒学
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分子生物学
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免疫学
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动物健康
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DOI
PubMed
高影响因子
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