斑马鱼蛋白酪氨酸硫酸化修饰的检测方法研究

doi:10.16288/j.yczz.22-018

遗传 ›› 2022, Vol. 44 ›› Issue (2): 178-186.doi: 10.16288/j.yczz.22-018

• 技术与方法 • 上一篇

斑马鱼蛋白酪氨酸硫酸化修饰的检测方法研究

张婷婷^1,²(), 刘峰²()

1. 中国科学技术大学生命科学与医学部,合肥 230026
2. 中国科学院动物研究所膜生物学国家重点实验室,中国科学院大学,北京 100101

收稿日期:2022-01-23 修回日期:2022-02-02 出版日期:2022-02-20 发布日期:2022-02-09
通讯作者: 刘峰 E-mail:zhangtingting@ioz.ac.cn;liuf@ioz.ac.cn
作者简介:张婷婷,在读硕士研究生,专业方向：生物工程。E-mail: zhangtingting@ioz.ac.cn
基金资助:
国家重点研发计划编号(2018YFA0800200);国家重点研发计划编号(2018YFA0801000);国家自然科学基金委重点项目编号(32030032);国家自然科学基金委重点项目编号(31830061);中国科学院战略性先导科技专项资助编号(XDA16010207)

Study on a detection method of protein tyrosine sulfation modification in zebrafish

Tingting Zhang^1,²(), Feng Liu²()

1. Division of Life Sciences and Medicine, University of Science and Technology of China, Hefei 230026, China
2. State Key Laboratory of Membrane Biology, Institute of Zoology, Chinese Academy of Sciences, University of Chinese Academy of Sciences, Beijing 100101, China

Received:2022-01-23 Revised:2022-02-02 Online:2022-02-20 Published:2022-02-09
Contact: Liu Feng E-mail:zhangtingting@ioz.ac.cn;liuf@ioz.ac.cn
Supported by:
Supported by the National Key Research and Development Program of China Nos(2018YFA0800200);Supported by the National Key Research and Development Program of China Nos(2018YFA0801000);the National Natural Science Foundation of China Nos(32030032);the National Natural Science Foundation of China Nos(31830061);the Strategic Priority Research Program of the Chinese Academy of Sciences, China No(XDA16010207)

摘要/Abstract

摘要：

蛋白酪氨酸硫酸化(protein tyrosine sulfation, PTS)是一种重要的翻译后修饰,调控生命活动中多种生理和病理过程,但由于PTS状态不稳定且目前缺乏有效的富集方法,因此在生物样品中难以进行有效地检测。本研究以模式动物斑马鱼(Danio rerio)为研究材料,利用Orbitrap Exploris 480高分辨质谱仪检测了斑马鱼胚胎发育早期总蛋白的酪氨酸硫酸化修饰水平,通过该方法共计检测到26种蛋白(包括膜蛋白、分泌蛋白、胞质蛋白和核蛋白等)存在潜在的29个酪氨酸硫酸化修饰位点。本研究建立了斑马鱼胚胎发育早期蛋白酪氨酸硫酸化修饰的检测方法,为探索生物体蛋白硫酸化修饰的作用机制奠定了技术基础。

关键词: 斑马鱼, 蛋白酪氨酸硫酸化, 高分辨质谱

Abstract:

Protein tyrosine sulfation (PTS) is an important post-translational modification that regulates a variety of physiological and pathological processes. However, PTS is unstable and lacks effective enrichment methods, which make it difficult to be detected in biological samples. In this study, we detected the tyrosine sulfation modification level of total proteins in developing zebrafish embryos by using high-resolution mass spectrometer, Orbitrap Exploris 480. A total of 26 proteins with tyrosine sulfation were detected, including membrane proteins, secreted proteins, cytoplasmic proteins and nucleoproteins. This study established a methodology in detecting protein tyrosine sulfation modification in embryonic zebrafish, which paved the way for evaluating the biological function of PTS.

Key words: zebrafish, protein tyrosine sulfation, high resolution mass spectrometry

张婷婷, 刘峰. 斑马鱼蛋白酪氨酸硫酸化修饰的检测方法研究[J]. 遗传, 2022, 44(2): 178-186.

Tingting Zhang, Feng Liu. Study on a detection method of protein tyrosine sulfation modification in zebrafish[J]. Hereditas(Beijing), 2022, 44(2): 178-186.

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试剂名称	公司	货号
碳酸氢铵(NH₄HCO₃)	Aladdin (北京)	A110537
氯化铵(NH₄Cl)	Aladdin (北京)	A116369
尿素(Urea)	Sigma-Aldrich (美国)	33247-1KG
碘乙酰胺(IAA)	Sigma-Aldrich (美国)	I1149-5G
三氟乙酸(TFA)	Sigma-Aldrich (美国)	T6508
乙腈(ACN)	Fisher (美国)	A955-4
甲酸(FA)	Fisher (美国)	A117-50
乙酸(AcOH)	Fisher (美国)	A35-500
甲醇(MeOH)	Fisher (美国)	A456-4
二硫苏糖醇(DTT)	Nacalai (北京)	14112-81
测序级胰蛋白酶(sequencing grade modified trypsin)	Promega (美国)	V5111
考马斯亮蓝G-250	Solarbio (广州)	C8420
BCA蛋白浓度测定试剂盒	Beyotime (上海)	P0012
弱阴离子交换柱(weak anion exchange column)	Waters (美国)	186002489
Empore固相萃取膜片	3M (美国)	2215-C18
无水乙醇	中国医药集团有限公司(北京)	10009218

仪器名称	公司	型号
非接触式超声破碎仪	戴杰德(比利时)	Diagenode Bioruptor Plus
隔膜真空泵	津腾实验设备有限公司(天津)	GM-0.33A
离心浓缩仪	Labconco (美国)	CentriVap
高分辨质谱仪	Thermo Scientific (美国)	Orbitrap Exploris 480

溶液名称	配方
考马斯亮蓝染液配方(200 mL)	0.4 g考马斯亮蓝G-250,90 mL无水乙醇和20 mL乙酸,用双蒸水定容至200 mL
考马斯亮蓝脱色液配方(500 mL)	250 mL无水乙醇和50 mL乙酸,用双蒸水定容至500 mL

UniProt数据库登录号	蛋白名称	肽段序列中的酪氨酸硫酸化修饰位点
E7F6T0	CD44a	AGELCQSLG-Y(SO₃)-R
Q90ZL2	Lancl1	TGYL-Y(SO₃)-SLIFVNQQFQQEK
Q5RHM3	Adgrf3b	VTITCSIFNSTET-Y(SO₃)-K
F1QPK1	Myof	IPGAKTADASDTGNAEGWE-Y( SO₃)-SSLIGWKFHR
Q6NSN2	Mao	MTANA-Y( SO₃)-DVIVIGGGISGLSAAK
A0A0H2UKT5	Ptprfa	HNTEDSSLTTISGLVPDIT-Y(SO₃)-GLR
E9QJI4	Si:ch211-264f5.6	LNGTALNSSQQV-Y(SO₃)-VVK
Q5U3G0	Pgrmc1	LLKPGEEPTE-Y(SO₃)-TDDEEVKDK
A0A0R4IRS1	Ap1m1	Y(SO₃)-ITQNGD-Y(SO₃)-QLR
A0A0R4INX3	Calua	EHDDGTNFE-Y(SO₃)-DHDAFLGEEEAK
A2CEA7	Rasgrf2	NCNPPCVP-Y(SO₃)-LGMYLTDLAFIEEGTPNFTEEGLVNFSK
A8WGQ0	Serpine1	NLALSPYGIASVLGMAQMGA-Y( SO₃)-GATLK
Q8AYE3	Serpinc1	LFGDKSTTFNETFQHISETV-Y( SO₃)-GAK
F1QVS8	Mybpc3	QLEV-Y(SO₃)-QSIADLTVK
A0A068FPN9	Ifit17c	FLQSL-Y(SO₃)-VLK
F2YWP2	Ttll10	FRAG-Y(SO₃)-QLL-Y(SO₃)-QIPNNK
G1K2X0	Ttn.1	SATKDQMTIEWNEPVIDGGSSVIG-Y(SO₃)-HLESK
B8A4F1	Si:dkey-15f17.8	TLTQNSDLISGV-Y(SO₃)-EGGLK
A0A0R4IW46	si:ch211-278p9.2	ELDLS-Y(SO₃)-NNPR
Q66I37	Nucks1a	KVVN-Y(SO₃)-SQFNESDDADEDYGRK
Q19AW4	Nfatc1	LALHPKP-Y(SO₃)-Y(SO₃)-SPPAMTPLMPTELGPCVTGPFASSPQR
A1A5H6	Cnot1	MNLDSLSLALSQIS-Y(SO₃)-LVDNLTKK
A1A605	Ybx1	DYQEN- Y(SO₃)-QSDPEAEPR
A3QK06	P2rx1	TFSNFFFE-Y(SO₃)-ETPR
F1QMN6	Spen	VNTSEGVVVLS-Y(SO₃)-SGQK
E9QD85	Rprd1b	VQEEEEEEDDDYRGH-Y(SO₃)-SPR

斑马鱼蛋白酪氨酸硫酸化修饰的检测方法研究

Study on a detection method of protein tyrosine sulfation modification in zebrafish

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