Glycoprotein Identification using Cleavable Bifunctional Probes★

doi:10.6023/A23050263

Acta Chimica Sinica ›› 2023, Vol. 81 ›› Issue (12): 1673-1680.DOI: 10.6023/A23050263 Previous Articles Next Articles

Special Issue: 庆祝《化学学报》创刊90周年合辑

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基于可断裂双功能探针的糖蛋白分析^★

李畅, 郑振东, 郑江南^*(), 田瑞军^*()

南方科技大学理学院化学系深圳 518055

投稿日期:2023-06-01 发布日期:2023-08-14
作者简介:
★庆祝《化学学报》创刊90周年.
基金资助:
项目受国家重点研发计划(2021YFA1302603); 项目受国家重点研发计划(2020YFE0202200); 国家自然科学基金(22125403); 国家自然科学基金(92253304); 国家自然科学基金(32201218); 深圳市科技创新委员会(JCYJ20200109141212325); 深圳市科技创新委员会(JCYJ20210324120210029); 深圳市科技创新委员会(JCYJ20200109140814408); 广东省杰出青年基金(2019B151502050)

Glycoprotein Identification using Cleavable Bifunctional Probes^★

Chang Li, Zhendong Zheng, Jiangnan Zheng(), Ruijun Tian()

Department of Chemistry, School of Science, Southern University of Science and Technology, Shenzhen 518055

Received:2023-06-01 Published:2023-08-14
Contact: *E-mail: zhengjn@sustech.edu.cn;tianrj@sustech.edu.cn
About author:
★Dedicated to the 90th anniversary of Acta Chimica Sinica.
Supported by:
National Key R&D Program of China(2021YFA1302603); National Key R&D Program of China(2020YFE0202200); National Natural Science Foundation of China(22125403); National Natural Science Foundation of China(92253304); National Natural Science Foundation of China(32201218); Shenzhen Innovation of Science and Technology Commission(JCYJ20200109141212325); Shenzhen Innovation of Science and Technology Commission(JCYJ20210324120210029); Shenzhen Innovation of Science and Technology Commission(JCYJ20200109140814408); Guangdong Provincial Fund for Distinguished Young Scholars(2019B151502050)

Glycoproteins play important roles in cellular activities and disease development. However, the abundance of glycoproteins is often low in complex biological samples. Thus, it is critical to enrich glycoproteins to achieve highly sensitive mass spectrometry (MS) analysis result. Many methods have been developed to enrich glycoproteins, among which hydrazide chemistry and oxime click chemistry have received increasing attention because of their universality and unbiasedness for labelling glycoproteins. Glycoproteins are captured by covalent binding or high affinity biotin binding on beads in these methods. Therefore, it is difficult to release glycoproteins from beads. Conventional way such as on-bead digestion is too harsh that can co-elute non-specifically bound proteins, endogenously biotinylated proteins and streptavidin on beads. These contaminant proteins would cause background interference in the subsequent glycoprotein identification by MS. In this study, three kinds of cleavable bifunctional probes named Biotin-Azo-Aminooxy (BAA), Biotin-Dde-Aminooxy (BDA) and Biotin-Nbz-Aminooxy (BNA) have been designed and synthesized. The cleavable bifunctional probes allow the release of glycoproteins from beads under mild condition. The mild condition can separate labelled glycoproteins from contaminant proteins to exclude the interference of non-glycoproteins. We evaluated the labelling and enrichment conditions, cleavage efficiency, glycoprotein recovery of these probes. The result showed that the properties of BDA and BNA are excellent. Lastly, BNA was selected to analyze cell surface glycoproteins by MS. Compared with the traditional method of on-bead digestion, the number of non-glycoproteins in this method decreased from 3564 to 2139 by 40.0% and the total Label-free quantitative (LFQ) intensity of glycoproteins increased by 104.2%. Furthermore, the endogenously biotinylated proteins were greatly reduced in cleavage method. The result shows that cleavable bifunctional probes can significantly improve the sensitivity and selectivity of the cell surface glycoprotein identification by MS, which provide tools for deep profiling of glycoproteins in the field of biology and medicine.

Key words: glycoprotein, mass spectrometry, enrichment, oxime click chemistry, cleavable probes

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Chang Li, Zhendong Zheng, Jiangnan Zheng, Ruijun Tian. Glycoprotein Identification using Cleavable Bifunctional Probes^★[J]. Acta Chimica Sinica, 2023, 81(12): 1673-1680.

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Fig. & Tab. 6

Figure 1. Workflow of cell surface glycoprotein identification using cleavable bifunctional probes

Scheme 1. The synthesis of the bifunctional probe BA and cleavable bifunctional probes BAA, BDA, BNA

Figure 2. (A) Labelling efficiency of the probes. Detection of the signal of biotin in labelled Fetuin by western blot (WB). (B) Enrichment effect of labelled protein. Enrich labelled Fetuin by Streptavidin beads and detect the Fetuin in supernatant and beads by Coomassie blue. Left lane: Labelled Fetuin. Middle lane: supernatant after capture with Streptavidin beads. Right lane: boiled beads after capture

Figure 3. (A) The best cleavage time and cleavage efficiency of different probes. Treat Fetuin labelled by different cleavable probes with different condition (BAA-50 mmol/L Na2S2O4; BDA-2% N2H4; BNA-365 nm UV) for 10 min, 30 min and 60 min. Detection of the remaining biotin in fetuin. (B) The recovery of labelled protein. Evaluation of the elution of Fetuin from Streptavidin beads. Left lane: boiled beads after capture. Middle lane: supernatant after cleavage and elution. Right lane: boiled beads after cleavage and elution

Figure 4. (A) Investigation of the gentleness of cutting conditions. Incubate Streptavidin beads with cell lysate. (B) Determination of protein recovery in cell lysate. Determination of protein recovery in cell lysate. Lane 1: 4 μg cell lysate. Lane 2: 4 μg cell lysate with 0.04 μg labelled Fetuin. Lane 3: 4 μg labelled Fetuin. Lane 4: Supernatant after cleavage and elution. Lane 5: boiled beads after cleavage and elution

Figure 5. Label the cell surface glycoproteins by cleavable bifunctional probe and pull down it by Streptavidin beads. Take On-bead digestion as control group and take cleavage as experimental group. (A) The number of non-glycoproteins and the ratio of glycoproteins in different groups; (B) the total LFQ intensity of glycoproteins in different groups; (C) the MS/MS counts of endogenously biotinylated proteins (such as MCCC1, ACACA, PCCA) in different groups

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基于可断裂双功能探针的糖蛋白分析^★

Glycoprotein Identification using Cleavable Bifunctional Probes^★

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基于可断裂双功能探针的糖蛋白分析★

Glycoprotein Identification using Cleavable Bifunctional Probes★

RichHTML

PDF

Knowledge

Abstract

Cite this article

share this article

Fig. & Tab. 6

References 29

Related Articles 15

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基于可断裂双功能探针的糖蛋白分析^★

Glycoprotein Identification using Cleavable Bifunctional Probes^★