基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法

doi:10.6023/A21010025

化学学报 ›› 2021, Vol. 79 ›› Issue (5): 663-669.DOI: 10.6023/A21010025 上一篇下一篇

研究论文

基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法

杨超^a^,^b, 单亦初^a^,^*(), 张玮杰^a^,^b, 戴忠鹏^a, 张丽华^a^,^*(), 张玉奎^a

a 中国科学院大连化学物理研究所中国科学院分离分析化学重点实验室大连 116023
b 中国科学院大学北京 100049

投稿日期:2021-01-27 发布日期:2021-03-30
通讯作者: 单亦初, 张丽华
基金资助:
项目受国家重点研发计划课题(2017YFF0205404); 项目受国家重点研发计划课题(2017YFA0505004); 国家自然科学基金(21675153); 国家自然科学基金(21725506)

Full-length Protein Sequencing Based on Continuous Digestion Using Non-specific Proteases

Chao Yang^a^,^b, Yi-Chu Shan^a^,^*(), Wei-Jie Zhang^a^,^b, Zhong-Peng Dai^a, Li-Hua Zhang^a^,^*(), Yu-Kui Zhang^a

a CAS Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Dalian 116023, China
b University of Chinese Academy of Sciences, Beijing 100049, China

Received:2021-01-27 Published:2021-03-30
Contact: Yi-Chu Shan, Li-Hua Zhang
About author:
*E-mail: shanyichu@dicp.ac.cn
E-mail:lihuazhang@dicp.ac.cn
Supported by:
Ministry of Science and Technology of China(2017YFF0205404); Ministry of Science and Technology of China(2017YFA0505004); National Natural Science Foundation of China(21675153); National Natural Science Foundation of China(21725506)

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1. Supporting Information-A21010025.pdf(300KB)

摘要/Abstract

对蛋白质全序列进行测定, 有助于分析蛋白质的结构, 揭示蛋白质的生物学功能. 针对目前基于质谱的蛋白质测序流程中使用特异性蛋白酶酶解产生的肽段种类少、重叠度低、序列拼接困难等问题, 发展了一种基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法. 构建了连续酶解装置, 并使用多种非特异性蛋白酶对蛋白质进行连续酶解. 利用非特异性蛋白酶酶解位点的非特异性、不同的酶解时间以及不同种类蛋白酶酶解产生肽段的互补性, 提高蛋白质酶解肽段的种类和重叠度, 并发展了蛋白质序列拼接算法对液相色谱质谱联用(LC-MS/MS)和从头测序获得的肽段序列进行拼接. 将此方法应用于牛血清白蛋白和单克隆抗体赫赛汀的全序列测定, 在不考虑亮氨酸和异亮氨酸的情况下, 对牛血清白蛋白和赫赛汀轻链的测序准确度达到100%, 赫赛汀重链的测序准确度为99.7%.

关键词: 非特异性蛋白酶, 连续酶解, 序列拼接, 全序列测定

Determining the complete sequence of the protein is helpful to analyze the structure of the protein and reveal the biological function of the protein. In traditional “bottom-up” proteomic strategy, database searching is used to identify sequences of peptides and proteins analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). It is impossible to identify proteins with unknown sequences through database searching, so de novo sequencing is essential for protein characterization. To increase the accuracy and coverage of protein sequencing, a de novo protein sequencing method based on continuous digestion using various non-specific proteases has been developed. A continuous digestion device was constructed, and a variety of non-specific proteases were used to continuously digest the protein. Taking advantage of the non-specific cleavage sites of non-specific proteases, the complementarity of peptides produced at different time and by different kinds of proteases, the type and overlapping degree of digested peptides were improved. The sequence coverage of peptides after continuous digestion by each protease can reach 100%. Finally, a sequence assembly algorithm was developed to assemble the peptides obtained by de novo sequencing. At first, the candidate peptide sequences were splitted into sequence tags which contain 7 amino acids, and then the most frequently occurring sequence tag was chosen as the seed sequence. Afterwards, the seed sequence was automatically or manually extended to the N-terminal end and C-terminal end respectively according to the scores of sequence tags. Finally, the complete protein sequence was successfully assembled. The developed method was applied to the de novo sequencing of bovine serum albumin (BSA) and monoclonal antibody Herceptin. Excluding leucine and isoleucine, full-length de novo sequencing was achieved with 100% accuracy for BSA and Herceptin light chain. Accuracy of the sequenced Herceptin heavy chain was 99.7%. The de novo sequencing strategy based on continuous digestion of proteins using non-specific proteases can be applied to de novo sequencing of proteins with unknown sequences or quality control of monoclonal antibody drugs.

Key words: non-specific protease, continuous digestion, sequence assembly, full-length sequencing

引用此文

杨超, 单亦初, 张玮杰, 戴忠鹏, 张丽华, 张玉奎. 基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法[J]. 化学学报, 2021, 79(5): 663-669.

Chao Yang, Yi-Chu Shan, Wei-Jie Zhang, Zhong-Peng Dai, Li-Hua Zhang, Yu-Kui Zhang. Full-length Protein Sequencing Based on Continuous Digestion Using Non-specific Proteases[J]. Acta Chimica Sinica, 2021, 79(5): 663-669.

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图/表 8

蛋白酶	时间1	时间2	时间3	时间4	时间5
菠萝蛋白酶	20	90	180	270	360
糜蛋白酶	20	90	150	300	420
弹性蛋白酶	20	120	180	240	420
蛋白酶K	20	90	180	270	360
链霉蛋白酶	20	40	120	150	180
嗜热菌蛋白酶	5	90	210	300	360

表1. BSA由不同蛋白酶酶解的时间(min)

Table 1. The digestion time (min) for BSA to be digested by different proteases

图1. BSA经过不同蛋白酶在不同酶解时间后所产生的肽段种类. (a)菠萝蛋白酶. (b)糜蛋白酶. (c)弹性蛋白酶. (d)蛋白酶K. (e)链霉蛋白酶. (f)嗜热菌蛋白酶

Figure 1. Number of BSA peptides produced using different proteases for different digestion times. (a) bromelain. (b) chymotrypsin. (c) elastase. (d) proteinase K. (e) pronase. (f) thermolysin

图2. 不同酶解时间所产生肽段被鉴定到的次数与数目

Figure 2. The frequencies and numbers of identified peptides produced at different digestion times

图3. 一次酶解和连续酶解方式所产生的肽段数目

Figure 3. Number of peptides produced by using one-time and continuous digestion

酶解方式	菠萝蛋白酶	糜蛋白酶	弹性蛋白酶	蛋白酶K	链霉蛋白酶	嗜热菌蛋白酶
一次酶解	100	94.34	98.63	96.23	96.91	97.77
连续酶解	100	100	100	100	100	100

表2. 连续酶解与一次酶解的序列覆盖率(%)

Table 2. Sequence coverage of continuous digestion and one-time digestion (%)

图4. 从头测序肽段的序列覆盖度

Figure 4. Sequence coverage of de novo sequencing peptides

图5. 序列拼接流程

Figure 5. The diagram of sequence assembly

图6. 序列拼接算法

Figure 6. Sequence assembly algorithm

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基于非特异性蛋白酶连续酶解的蛋白质全序列测定方法

Full-length Protein Sequencing Based on Continuous Digestion Using Non-specific Proteases

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