基于可断裂双功能探针的糖蛋白分析★

doi:10.6023/A23050263

化学学报 ›› 2023, Vol. 81 ›› Issue (12): 1673-1680.DOI: 10.6023/A23050263 上一篇下一篇

所属专题：庆祝《化学学报》创刊90周年合辑

研究论文

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)

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摘要/Abstract

糖蛋白在细胞活动和疾病发生发展过程中发挥着重要作用. 然而, 在复杂的生物样本中, 糖蛋白的丰度往往较低, 因而在质谱分析前必须对其进行富集以实现高灵敏质谱分析. 目前, 已经发展出诸多糖蛋白富集方法, 其中肼化学法与肟点击化学法因对糖蛋白标记的普适性与无偏性受到了越来越多的关注. 然而, 由于这些方法以共价连接或者超高亲和力生物素结合的方式在固相载体上捕获糖蛋白, 因此无法选择性地洗脱被富集的糖蛋白. 传统的方法是在固相载体上直接进行酶解, 因此无法排除非特异性吸附蛋白、内源性生物素化蛋白、固相载体上的链霉亲和素等无关蛋白对糖蛋白质谱分析产生的背景干扰. 本工作设计并合成了三种可断裂的双功能探针[生物素-偶氮苯-氧胺(BAA)、生物素-N-1-(4,4-二甲基-2,6-二氧环亚己基)乙基-氧胺(BDA)、生物素-邻硝基苄基-氧胺(BNA)], 通过温和的方式从固相载体上释放糖蛋白, 从而排除非糖蛋白的干扰, 提高糖蛋白富集的选择性. 对三种探针的标记与富集效果、切割效率、蛋白回收率等性能进行了评估. 结果显示, 探针BDA与BNA的性能较为优越. 最后, 探针BNA被应用于细胞表面糖蛋白的分离富集与质谱鉴定中. 与传统的在固相载体上直接进行酶解的方法相比, 该方法中非糖蛋白数目从3564降到了2139, 降幅达到了40.0%, 糖蛋白的无标定量(LFQ)分析强度提升了104.2%. 此外, 切割方法极大降低了内源性生物素化蛋白的干扰. 上述结果表明, 可断裂双功能探针显著地提高了糖蛋白质谱鉴定的灵敏度与选择性, 为生物与医学等领域中糖蛋白的深度解析提供了新的工具.

关键词: 糖蛋白, 质谱, 富集, 肟点击化学, 可断裂探针

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

引用此文

李畅, 郑振东, 郑江南, 田瑞军. 基于可断裂双功能探针的糖蛋白分析^★[J]. 化学学报, 2023, 81(12): 1673-1680.

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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图/表 6

图1. 基于可断裂双功能探针的细胞表面糖蛋白分析流程图

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

图式1. 双功能探针BA及可断裂双功能探针BAA、BDA、BNA的合成

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

图2. (A)探针的标记效果. 以蛋白免疫印迹法(WB)检测标记后Fetuin上生物素的信号. (B)标记蛋白的富集效果. 以链霉亲和素琼脂糖微球富集标记探针后的Fetuin. 以考马斯亮蓝染色法检测富集后上清与微球中蛋白的总量. 左侧泳道: 标记探针后的Fetuin; 中间泳道: 与链霉亲和素琼脂糖微球孵育后的上清; 右侧泳道: 孵育后煮样的微球

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

图3. (A)探针的最佳切割时间与探针的切割效率. 以切割条件BAA-50 mmol/L Na2S2O4 ; BDA-2% N2H4; BNA-365 nm UV分别对标记了探针的Fetuin处理10 min、30 min、60 min, 然后检测胎球蛋白上剩余的生物素含量. (B)标记蛋白的回收率. 对微球进行切割及洗脱条件处理后, 检测上清与微球中蛋白的量. 左侧泳道: 孵育后直接煮样的微球; 中间泳道: 对孵育后的微球进行切割与洗脱处理后收集合并的上清; 右侧泳道: 孵育后进行切割与洗脱处理后煮样的微球

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

图4. (A)切割条件的温和性考察. 将链霉亲和素琼脂糖微球与细胞裂解液孵育. (B)细胞裂解液中蛋白的回收率测试. 在400 μg的细胞裂解液中加入4 μg标记探针的Fetuin, 加入链霉亲和素琼脂糖微球孵育. 第1泳道: 细胞裂解液(4 μg); 第2泳道: 加入0.04 μg标记的Fetuin的4 μg细胞裂解液; 第3泳道: 标记探针的Fetuin (4 μg); 第4泳道: 对微球进行切割洗脱后的上清; 第5泳道: 进行切割与洗脱处理后煮样的微球

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

图5. 将细胞表面糖蛋白用可断裂双功能探针进行标记, 并用链霉亲和素琼脂糖微球富集进行富集. 以传统的On-bead digestion方法为对照组, 以切割洗脱的方法为实验组. 在两组实验组鉴定到的(A)非糖蛋白的数目以及糖蛋白的占比; (B)糖蛋白总的无标定量分析强度对比; (C)内源性生物素化蛋白(如MCCC1、ACACA、PCCA)的MS/MS数目对比

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★

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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^★