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瑕瑜研究组-自由基生物质谱

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Welcome

Our research program aims broadly at developing new mass spectrometry (MS) methods for bio-analysis. Research efforts are currently focused on utilizing radical reactions as a unique approach to providing the high level of structural information of proteins and lipids, such as disulfide linkage pattern and carbon-carbon double bond location. We are also developing new MS instrumentation to investigate the radical attack on biomolecules in the gas phase and characterizing a series of peptide or protein radicals which are of biological importance. 

TLR天然免疫信号传递中PI4P介导的细胞外囊泡生成新机制

细胞外囊泡(Extracellular Vesicles,EVs)是细胞分泌的磷脂双分子层膜包裹而成的小泡(图1)。1996年首次在抗原呈递过程中发现,它不仅是细胞排出垃圾的方式,更在细胞间信号传递中发挥重要功能。Toll样受体(Toll-like receptor, TLR)是天然免疫与适应性免疫之间最重要的桥梁之一,严格的信号传导和时空调控为免疫系统的有序运行提供保障,可有效避免脓毒症等天然免疫疾病的发生。然而,EVs作为重要的细胞间通讯介质,在TLRs信号传递过程中的功能报道尚少,同时TLRs激活是否会调控EVs的生成与释放尚无充分证据。

2023年4月,来自清华大学的尹航研究组在Journal of Extracellular Vesicles上发表题为“Exosomal lipid PI4P regulates small extracellular vesicle secretion by modulating intraluminal vesicle formation”的文章,报道了一项新的PI4P在调控EV生成的分子机制,并且为EVs调控天然免疫TLR4信号调控提供新证据,为新的EV调控与TLR4天然免疫信号调控提供潜在靶点(图1)。

图1 LPS-TLR4 信号通路调节 EVs 释放的分子机制模式图

该研究发现,在TLR4激活条件下,巨噬细胞内EVs释放速率随TLR4的配体革兰氏阴性细菌外部细胞壁的一种主要糖脂组分——脂多糖(Lipopolysaccharide,LPS)刺激时间先升高后降低,表现出明显的时间规律。通过与清华大学化学系瑕瑜课题组合作,成功实现对EVs中微量磷脂——磷脂酰肌醇的定量检测,发现介导囊泡生成的磷脂酰肌醇-4-磷酸(Phosphatidylinositol-4-phosphate,PI4P)与EVs的释放速率呈显著正相关关系(图2)。

图2 LPS刺激调控EV释放速率变化及EV中PIP含量

探究分子机制发现,LPS刺激短时间内,TLR4激活产生的I型干扰素可调控PI4P激酶表达增加多囊泡体(MVB)上PI4P含量,从而招募下游蛋白促进胞内囊泡(ILV)生成,从而增加EV释放速率;LPS刺激时间增长后,PI4P从MVB减少,从而EVs生成与释放减少。TLR4受体持续性激活所致的过度免疫反应是脓毒血症的主要诱因,EVs所参与的严格时序调控将有效避免信号的持续性激活。

进一步探究EVs释放与LPS刺激信号的关系,发现TLR4天然免疫信号激活后,巨噬细胞内LPS-TLR4下游的TRIF信号通路激活所产生 IFN-β,对PI(4,5)P2激酶PIP5K1C的表达调控实现,通过改变PI4P的定位调控EV的生成(图3)。该结论在巨噬细胞RAW264.7细胞系和C57BL/6J小鼠原代BMDMs生理模型中均表现出一致的调控作用。

图3  LPS-IFN-β信号通路调控PIP变化及EV前体的生成

近年来,由于EV可以实现液体活检以及载药透过血脑屏障等优势,该领域的研究持续升温。天然免疫中EV调控的新机制的发现为疾病的发病机理提供新解释,同时EV生成机制的解析也为其标准化生产提供潜在参考途径。

 

本文编辑:夏天

本文审核:靳学

原文链接:https://doi.org/10.1002/jev2.12319

鞘氨醇-1-磷酸转运蛋白(SPNS2)转运机制研究

哈工大生命科学中心何元政课题组在鞘氨醇-1-磷酸(S1P)通过人鞘氨醇-1-磷酸转运蛋白(SPNS2)转运的结构基础方面取得新进展,揭示了SPNS2独特的S1P转运机制。12月20日,研究成果以《鞘氨醇-1-磷酸通过人鞘氨醇-1-磷酸转运蛋白(SPNS2)转运的结构基础》(Structural basis of Sphingosine-1-phosphate transport via human SPNS2)为题发表在《细胞研究》(Cell Research)上。该研究阐明了SPNS2介导的S1P转运,并有助于开发新型SPNS2抑制剂,为治疗自身免疫疾病提供新的靶点。

鞘氨醇-1-磷酸是细胞膜鞘脂的代谢物,作为脂质信号分子在免疫反应、血管发育和神经系统稳态等多种生理过程中发挥重要作用。S1P的适当空间梯度是S1P信号转导的关键,S1P空间梯度的建立是由S1P转运蛋白(包括SPNS2和MFSD2B)将S1P从细胞内侧运输到细胞外侧来实现的。尽管在建立S1P的空间梯度中起着关键作用,但目前对S1P的转运机制知之甚少。

基于在S1P转运中发挥的重要作用和靶向SPNS2降低自身免疫性疾病方面的可能性,何元政课题组运用冷冻电镜技术解析了SPNS2的无配体结合状态、S1P结合状态、FTY720-P结合状态和抑制剂16d结合的多种状态结构(图1)。该研究开发出抗SPNS2的纳米抗体,以克服小尺寸膜蛋白在冷冻电镜结构解析中的困难,并开发出S1P转运检测方法来评估SPNS2的转运活性。该研究揭示了SPNS2门控区域中由Y246和G333形成的氢键是控制底物转运的关键因素(图2)。该研究还提出了用“阶梯”模型来阐述SPNS2的底物转运机制,底物的磷酸头基是运用SPNS2细胞内腔中一系列极性残基形成的“梯子”爬升到达了门控区域,进而进行脂质转运(图2)。此外,该研究还解析了最新研发的SPNS2抑制剂16d结合SPNS2的结构,并对其抑制机理进行了阐释。综上所述,通过解析SPNS2及其与小分子复合物的结构,该研究为理解S1P的转运机制提供了框架,并为设计靶向SPNS2的抑制剂提供了结构基础。

图1. SPNS2及其与小分子复合物的冷冻电镜结构

图2. SPNS2的底物转运机制

何元政课题组博士研究生段亚宁、新加坡国立大学医学院生物化学系阮龙(Long N.Nguyen)课题组博士研究生梁南希(Nancy C.P.Leong)、清华大学化学系瑕瑜课题组博士研究生赵婧为并列第一作者。何元政研究员、阮龙教授为共同通讯作者。何元政课题组硕士研究生张羽,博士研究生王娜,博士后徐珍媚,博士研究生夏瑞雪、马正雄、钱雨、尹晗、祝鑫焱,阮龙课题组博士研究生阮达特(Dat T.Nguyen)、哈和(Hoa T.T.Ha),瑕瑜教授参与该课题相关研究工作。

该研究获国家自然科学基金和哈工大生命科学中心启动基金等资助。

 

本文编辑:梁英爽,段亚宁

本文审核:赵   婧

原文链接:https://doi.org/10.1038/s41422-023-00913-0

揭示油酸为孤儿受体(GPR3)的天然配体

哈工大生命科学中心何元政课题组联合陈政课题组、清华大学瑕瑜课题组在孤儿受体(GPR3)的结构研究中取得重要进展。研究成果以《油酸为GPR3的内源性配体》(Identification of oleic acid as an endogenous ligand of GPR3)为题发表在《细胞研究》(Cell Research)上。研究成果揭示了GPR3的天然配体、自激活机制和其在冷刺激诱导下的产热机理,为相关代谢类疾病治疗提供新思路。

GPR3作为A类G蛋白偶联受体中的孤儿受体,有较高的组成性活性,在神经系统和代谢方面发挥关键作用。在神经系统方面,GPR3可调节情绪,参与神经性疼痛和成瘾过程,且与阿尔茨海默症(老年痴呆)密切相关。在代谢方面,冷刺激下能诱导GPR3高表达并驱动脂肪产热等,因此具有治疗包括肥胖症和糖尿病等代谢类疾病的可能,然而其结构信息和内源性配体尚不明确。

何元政课题组运用冷冻电子显微镜解析了GPR3与Gs蛋白的复合物结构,发现其分辨率达2.79埃,同时在结构分析中发现GPR3的配体结合口袋中有脂质类配体的电子云密度,通过质谱分析鉴定出该密度为油酸(oleic acid, OA)分子(图1)。此后的结构和功能分析表明,GPR3内的疏水通道连接了受体的胞外侧与细胞膜中部,使细胞膜内的脂肪酸易于与受体结合(图1)。同时,油酸、棕榈酸、月桂酸等游离脂肪酸(free fatty acid, FFA)可以结合并激活GPR3,而溶血磷脂酸则不能。进一步的动物实验表明,冷刺激可诱导小鼠体内OA的分泌,进而激活棕色脂肪组织中的Gs/cAMP/PKA信号通路,而Gpr3基因敲除小鼠在冷刺激时则对OA无反应。对此,研究人员针对GPR3的激活机制提出了“生而激活,遇冷则强”(born to be activated and cold to enhance)模型(图2),为理解GPR3激活和冷刺激下的产热机制提供基础。

图一. GPR3/Gs复合物冷冻电镜结构和配体鉴定

图二. GPR3“生而激活,遇冷则强”模型

哈工大何元政研究员、陈政研究员,清华大学瑕瑜教授为论文共同通讯作者。何元政课题组博士研究生熊杨杰、博士后徐珍媚,陈政课题组博士后李新志、王玉琴博士,瑕瑜课题组博士研究生赵婧为论文并列第一作者。何元政课题组王娜博士和博士研究生段亚宁、夏瑞雪、钱雨、梁佳乐,哈工大生命科学中心韩正滨高级工程师,日本东北大学井上飞鸟教授参与该课题研究工作。

该研究获国家自然科学基金和哈工大生命科学中心启动基金资助。

 

本文编辑:梁英爽 熊杨杰

本文审核:赵   婧

原文链接:https://doi.org/10.1038/s41422-024-00932-5

1. Gas-phase radical ion chemistry

Radical ions, which consist of unpaired electrons, offer distinct gas-phase ion chemistry as compared to the even-electron species. Radical chemistry can be utilized to tackle challenging problems, such as differentiating isomeric structures, which would otherwise not be solved by traditional MS analysis of even-electron ions of the biomolecules. We are developing MS instrumentation and methods to facilitate radical reactions for either in the vacuum or in ambient air.

1. Radical reactions at the interface of ESI-MS.

Radicals or excited neutrals are generated via air discharge or UV photolysis and subsequently reacted with ions entrained in the ESI plume.  Radical reactions are subsequently monitored and characterized in situ by MS analysis. Reactions of peptides and lipids with various radical species have been investigated, including •OH, •CH2OH, excited state of (CH3)2CO.  Novel analytical applications based on these reactions have been developed.

AC; 2010, 82, 2856JASMS., 2011, 22, 922Analyst, 2013, 138, 2840;JASMS, 2014, 25, 1192.

 

2. Ion/radical reactions in a linear ion trap mass spectrometer.

A first linear ion trap mass spectrometer capable of studying reactions between the mass-selected ions and radicals has been recently developed and tested in collaboration with Prof. Zheng Ouyang from Biomedical Engineering at Purdue. This instrument uses a rectilinear ion trap as the mass analyzer and gas-phase reactor, an ESI as the source of the bimolecular ions, a pulsed pyrolysis valve for the generating an intense radical beam, and a glow discharge electron impact (GDEI) source for radical characterization. This MS platform can facilitate mechanistic studies on the radical attack to biomolecules that are of biological significance

 

3. Chemistry of bio-radical ions.

Through radical relations at ESI-MS interface, our group has synthesized and studied cysteine sulfinyl radical in the gas phase (Cys-SO•), which has a wide relevance to radical-induced oxidation of proteins, however, has been poorly characterized due to its transient nature in the condensed phases. Different from carbon-centered radicals, we have discovered that sulfinyl radical has a dual property of being acting as a base or a radical via combined experimental and theoretical approaches. The base property allows the formation of proton bridging between the radical site and the neighboring amino acid residues and thus contributes to the overall structural and chemical property of a polypeptide.

Love et al. J. Am. Chem. Soc., 2013, 135, 6226

Tan et al. J Phys. Chem. A, 2014, 118 ,11828

 

We also systematically investigated the inter- and intra-molecular reactivity of the sulfinyl radicals. They showed that the cysteine sulfinyl radical can react with a disulfide bond or a thiol group within a peptide, which has implications to radical-induced disulfide bond scrambling.

Using functionalized sulfinyl radical as a precursor of glycyl-type radical, we have also developed an experimental approach based on tandem mass spectrometry to correlate the electronic property of the connecting groups to the stability of glycyl-type radical species (Angew. Chem., Int. Ed., 2014, 53, 1887-1890, featured as the front cover and the “hot article”).

Tan et al. Angew. Chem. Int. Ed. 2014, 53, 1887

4. Application of radical chemistry for bio-analysis Analysis of unsaturated lipids:

Facile determination of C=C bond locations of lipids is a long-standing challenge for lipid analysis using MS. Intact lipid analysis via conventional low energy collisional activation tandem mass spectrometry does not provide information for the C=C location because much higher energies are required for cleaving C-C or C=C bonds and thus no fragments specific to the C=C locations can be produced. Utilizing the high reactivity of C=C with radicals or electrophilic excited state molecules, our group has recently developed coupling Paternò–Büchi (PB) reaction with MS/MS for highly confident C=C bond location determination in lipids (Angew. Chem., Int. Ed, 2014, 53, 2592-2596). This PB-MS/MS strategy is currently being developed for unsaturated lipid C=C location isomer characterization and quantitation of biological samples (tissue, cell lines, plasma), application to shotgun and separation based lipidomics, biomarker discovery, and bio-imaging.

Ma and Xia, Angew. Chem. Int. Ed. 2014, 53, 2592

 

2. PB-MS/MS developed by our group

42. Tian Xia, Xue Jin, Donghui Zhang, Jitong Wang, Ruijun Jian, Hang Yin, Yu Xia*, "Alternative fatty acid desaturation pathways revealed by deep profiling of total fatty acids in RAW 264.7 cell line", J. Lipid. Res. 2023.

https://doi.org/10.1016/j.jlr.2023.100410

41.  Qiaohong Lin, Ruijun Jian, Shengzhuo Wang, Yu Xia*, "Characterization of Oxidized Glycerophosphoethanolamines via Radical-Directed Dissociation Tandem Mass Spectrometry and the Paternò–Büchi Derivatization", Anal. Chem. 2023, 95, 25, 9422–9427.

https://doi.org/10.1021/acs.analchem.3c00792

40.  Tian Xia, Feng Zhou, Donghui Zhang, Xue Jin, Hengxue Shi, Hang Yin, Yanqing Gong, Yu Xia*, "Deep-profiling of phospholipidome via rapid orthogonal separations and isomer-resolved mass spectrometry", Nat. Commun., 202314, 4263.

https://doi.org/10.1038/s41467-023-40046-x

39.  Hengxue Shi, Zhenshu Tan, Xiangyu Guo, Hanlin Ren, Shengzhuo Wang, Yu Xia*, "Visible-Light Paternò–Büchi Reaction for Lipidomic Profiling at Detailed Structure Levels", Anal. Chem. 2023, 95, 11, 5117–5125.

https://doi.org/10.1021/acs.analchem.3c00085

38.  Wenpeng Zhang, Ruijun Jian, Jing Zhao, Yikun Liu, Yu Xia*, "Deep-lipidotyping by mass spectrometry: recent technical advances and applications", Journal of Lipid Research, 2022.

https://doi.org/10.1016/j.jlr.2022.100219

37.  Donghui Zhang, Qiaohong Lin, Tian Xia, Jing Zhao, Wenpeng Zhang, Zheng Ouyang* and Yu Xia*, "LipidOA: A Machine-Learning and Prior-Knowledge-Based Tool for Structural Annotation of Glycerophospholipids", Anal. Chem. 2022, 94, 48, 16759–16767.

https://doi.org/10.1021/acs.analchem.2c03505

36. Hai-Fang Li, Jing Zhao, Wenbo Cao, Wenpeng Zhang, Yu Xia*, and Zheng Ouyang*, “Site-Specific Photochemical Reaction for Improved C=C Location Analysis of Unsaturated Lipids by Ultraviolet Photodissociation” Research, 2022, Article ID 9783602, Published: 12 Feb 2022

https://doi.org/10.34133/2022/9783602

35. Xiaoxiao Ma, Wenpeng Zhang, Zishuai Li, Yu Xia* and Zheng Ouyang*, Enabling High Structural Specificity to Lipidomics by Coupling Photochemical Derivatization with Tandem Mass Spectrometry. Acc. Chem. Res. 2021, 54, 20, 3873–3882.

https://doi.org/10.1021/acs.accounts.1c00419

34. Zishuai Li, Simin Cheng, Qiaohong Lin, Wenbo Cao, Jing Yang, Minmin Zhang, Aijun Shen, Wenpeng Zhang, Yu Xia, Xiaoxiao Ma* and Zheng Ouyang*, "Single-cell lipidomics with high structural specificity by mass spectrometry" Nature Communications, 2021, 12, 2869.

https://doi.org/10.1038/s41467-021-23161-5

33. Qiaohong Lin, Pengyun Li, Mengxuan Fang, Donghui Zhang, and Yu Xia*, “Deep Profiling of Aminophospholipids Reveals a Dysregulated Desaturation Pattern in Breast Cancer Cell Lines” Anal. Chem. 2021, Publication Date:December 21

https://doi.org/10.1021/acs.analchem.1c03494

32. Jing Zhao, Mengxuan Fang, Yu Xia*, “A Liquid Chromatography-Mass Spectrometry Workflow for In-Depth Quantitation of Fatty Acid Double Bond Location Isomers”J. Lipid. Res. 2021, Available online 24 August .

https://doi.org/10.1016/j.jlr.2021.100110

31. Qingyuan Hu, Yu Xia*, Xiaoxiao Ma*, “Comprehensive Structural Characterization of Lipids by Coupling Paternò–Büchi Reaction and Tandem Mass Spectrometry”, In: Hsu FF. (eds) Mass Spectrometry-Based Lipidomics. Methods in Molecular Biology, vol 2306. Humana, New York, NY.

https://doi.org/10.1007/978-1-0716-1410-5_4

30. Tian Xia, Ming Yuan, Yongwei Xu, Feng Zhou, Kate Yu*, and Yu Xia*, “Deep Structural Annotation of Glycerolipids by the Charge-Tagging Paterno–Büchi Reaction and Supercritical Fluid Chromatography–Ion Mobility Mass Spectrometry", Anal. Chem. 2021, 93, 23, 8345–8353

https://pubs.acs.org/doi/10.1021/acs.analchem.1c01379

29. Hanlin Ren, Alexander Triebl, Sneha Muralidharan, Markus R. Wenk*, Yu Xia* and Federico Torta*, "Mapping the Distribution of Double Bond Location Isomers in Lipids across Mouse Tissues", Analyst, 2021,146, 3899-3907

https://doi.org/10.1039/D1AN00449B

28. X. Ma, Y. Xia, "Unsaturated Lipid Analysis via Coupling the Paternò–Büchi Reaction with ESI-MS/MS", Lipidomics. 2020: 148-174.

27. Xue Zhao, Gang Wu, Wenpeng Zhang, Mengqiu Dong, and Yu Xia*, "Resolving Modifications on Sphingoid Base and N-Acyl Chain of Sphingomyelin Lipids in Complex Lipid Extracts", Anal. Chem. 2020, 92, 21, 14775–14782

https://doi.org/10.1021/acs.analchem.0c03502

26. Jing Zhao, Xiaobo Xie, Qiaohong Lin, Xiaoxiao Ma, Pei Su, Yu Xia*, "Next-Generation Paternò–Büchi Reagents for Lipid Analysis by Mass Spectrometry", Anal. Chem. 2020, 92, 19, 13470–13477

https://pubs.acs.org/doi/10.1021/acs.analchem.0c02896

25. Elissia T. Franklin, Yu Xia*, "Structural elucidation of triacylglycerol using online acetone Paternò–Büchi reaction coupled with reversed-phase liquid chromatography mass spectrometry" , Analyst. 2020, 145, 6532-6540.

https://doi.org/10.1039/D0AN01353F

24. Elissia Franklin, Samuel Shields, Jeffrey Manthorpe, Jeffrey C. Smith, Yu Xia, Scott A. Mcluckey*, "Coupling Headgroup and Alkene Specific Solution Modifications with Gas-Phase Ion/Ion Reactions for Sensitive Glycerophospholipid Identification and Characterization", J. Am. Soc. Mass Spectrom. 2020, 31, 4, 938–945.

https://doi.org/10.1021/jasms.0c00001

23. Wenpeng Zhang*, Bing Shang, Zheng Ouyang, Yu Xia*, "Enhanced Phospholipid Isomer Analysis by Online Photochemical Derivatization and RPLC-MS", Anal. Chem. 2020, 92, 9, 6719–6726.

https://doi.org/10.1021/acs.analchem.0c00690

22. Tian Xia, Hanlin Ren, Wenpeng Zhang, Yu Xia*, "Lipidome-Wide Characterization of Phosphatidylinositols and Phosphatidylglycerols on C=C Location Level", Analytica Chimica Acta, 2020, 1128, 107-115.

http://dx.doi.org/10.1016/j.aca.2020.06.017

21. Wenbo Cao, Simin Cheng, Jing Yang, Wenpeng Zhang, Zishuai Li, Qinhua Chen, Yu Xia, Zheng Ouyang*, Xiaoxiao Ma*, "Large-scale lipid analysis with C=C location and sn-position isomer resolving power", Nat Commun, 2020, 11, 375.

https://www.nature.com/articles/s41467-019-14180-4

20. 马潇潇,胡清源,瑕瑜*, "Paternò-Büchi(PB)反应与串联质谱结合实现不饱和脂质精确结构解析", 分析测试学报, 2020, 39(1), 19-27.

http://new.fxcsxb.com/fxcsxb/ch/reader/view_abstract.aspx?file_no=20200103&flag=1

19. Xiaobo Xie, Jing Zhao, Miao Lin, Jinlan Zhang, Yu Xia*, "Profiling of Cholesteryl Esters by Coupling Charge Tagging Paternò-Büchi Reaction and Liquid Chromatography-Mass Spectrometry", Anal. Chem. 2020, 92, 12, 8487–8496.

https://doi.org/10.1021/acs.analchem.0c01241

18. Haifang Li, Wenbo Cao, Xiaoxiao Ma, Xiaobo Xie, Yu Xia, Zheng Ouyang*, "Visible-Light-Driven [2 + 2] Photocycloadditions between Benzophenone and C=C Bonds in Unsaturated Lipids", J. Am. Chem. Soc. 2020, 142, 7, 3499–3505.

https://doi.org/10.1021/jacs.9b12120

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13. Elissia T. Franklin, Stella K. Betancourt, Caitlin E. Randolph, Scott A. McLuckey* , and Yu Xia* ,"In-depth structural characterization of phospholipids by pairing solution photochemical reaction with charge inversion ion/ion chemistry",  Anal Bioanal Chem. 2019, 411, 4739–4749.

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37. Hynds H. M.;Hines K. M.*; Ion Mobility Shift Reagents for Lipid Double Bonds Based on Paternò–Büchi Photoderivatization with Halogenated Acetophenones. J. Am. Soc. Mass Spectrom. 2022, 33, 10, 1982–1989

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14. Wäldchen, F.; Spengler, B.; Heiles, S. "Reactive Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Using an Intrinsically Photoreactive Paternò–Büchi Matrix for Double-Bond Localization in Isomeric Phospholipids." Journal of the American Chemical Society, 2019, 141, 11816-11820.

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