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Li Huilin’s team achievement:non denaturing top-down mass spectrometry for structural characterization of proteins and their complexes

Hello, everyone. This week, I’d like to share with you a review recently published by lihuilin’s research group on mass spectroscopy reviews, native top ‐ down mass spectroscopy for higher ‐ order structural characterization of proteins and complexes1.

The rapid development of structural biology has greatly promoted the development of protein structure characterization tools. Among them, the analytical methods based on mass spectrometry stand out with its advantages of fast, sensitive and high-throughput. Compared with high-resolution structural characterization tools at the atomic level, such as X-ray crystallography, nuclear magnetic resonance (NMR), cryoelectron microscopy (cryo EM), etc., mass spectrometry based analysis methods can effectively supplement the information of protein dynamic structural changes, and are not limited by protein purity and molecular weight. Compared with low resolution protein characterization tools such as circular dichroism spectroscopy and dynamic light scattering, mass spectrometry based analysis methods can provide higher horizontal resolution of peptides or residues, and obtain additional sequence, post ‐ translational modifications (PTMs), local spatial structure and other information. Common structural mass spectra include:hydrogen deuterium exchange MS (hdx-ms), cross linking MS (cx-ms), surface labeling MS (cl-ms), etc. These methods have been introduced in detail in many literatures, 2,3 and will not be repeated here. This review will focus on the application of native top-down mass spectrometry (ntdms) in the structural characterization of proteins and their complexes.

In the past decade, non denaturing mass spectrometry (NMS), especially ntdms, has developed rapidly. As a bridge connecting proteomics and structural biology, NMS retains the characteristics of non covalent interaction, which makes it widely used in the characterization of protein complexes, such as deduced subunit composition, stoichiometric ratio, subunit arrangement, etc. However, for some deep-seated structural information, such as amino acid sequences, PTMs, ligand binding sites, subunit binding interfaces, etc., it is impossible to obtain only by a single NMS. Correspondingly, top-down mass spectrometry (TDMS) under denatured conditions can directly obtain sequence and PTMs information at the complete protein level. Although it is conducive to the accurate localization of PTM and the identification of protein and protein heterogeneity, it loses high-level structural information involving non covalent interactions. Limited by the development of mass spectrometers, in the early days, NMS and TDMS were usually carried out in two independent experiments. With the development of mass analyzers and various activation/fragmentation methods, NMS and TDMS can effectively combine and give full play to their respective advantages. While realizing the acquisition of multi-level structure information, they are also constantly challenging more complex biological systems, such as ribosomes, membrane proteins, endogenous protein mixtures, etc.

experimental design

Ntdms has become an important tool to characterize the primary to advanced structures of proteins and complexes. With the increase of the size and complexity of protein samples, instruments used in ntdms not only need to meet some specific standards, but also need to continuously improve their performance to meet these increased needs. Several key steps in ntdms analysis include sample pretreatment, ESI ionization, secondary fragmentation, quality detection and data processing.

Sample pretreatment

为了维持蛋白的自然状态,通常需要在生理环境中进行nMS分析。然而,缓冲液中的非挥发性盐会产生大量盐簇并与蛋白离子形成非特异性加合物,从而抑制离子信号、降低检测的准确度和灵敏度。因此,Sample pretreatment过程中最重要的环节就是除盐。然而适当的离子强度有助于维持蛋白的三维结构,所以通常的步骤是对蛋白进行缓冲液置换,将蛋白置换至醋酸铵或碳酸氢铵等挥发性盐溶液中。目前已开发了多种在线或离线的除盐方法,详细内容的可在综述原文中查看,此处不再赘述。除了使用非挥发性缓冲盐,减小ESI喷针孔径大小也可以提高系统耐盐能力。

Fragmentation/activation mode

Secondary fragmentation mode is the key to realize NMS to ntdms. Common activation methods can be divided into three categories according to the principle:collision based (CID, SID), electron based (ECD, ETD, Eid, etc.) and photon based (uvpd, IRMPD) activation/fragmentation methods. It is worth noting that both CID and IRMPD belong to the activation mode of slow heating, and the energy accumulation is very slow, so that the energy rearrangement has been carried out before fragmentation, and some weak or unstable bonds will preferentially break, eventually leading to the destruction of non covalent interaction in the process of activation. Sid, Exd and uvpd belong to the activation mode of fast heating. Fragmentation occurs before energy rearrangement, and non covalent interactions can be retained in this process. The degree of fragmentation is limited by non covalent interactions, so it can be used to characterize the spatial structure of proteins. In addition, the combination of multiple activation methods or series connection with ion mobility technology is also the key to obtain multi-level structure information.

Quality inspection

Compared with mass spectrometry analysis under denatured conditions, the number of charges generated by electrospray spray ionization of protein complexes in natural environment is relatively small, so a mass analyzer with a large m/z range (up to m/z=20000 Da or even higher) is required. Initially, NMS analysis was highly dependent on time of flight (TOF) based mass analyzers because TOF has a theoretically infinite m/z range. In recent years, high-resolution mass analyzers such as Orbitrap and Fourier transform ion cyclotron resonance (FTICR) have brought new vitality to ntdms analysis of biological macromolecules. In the review, we briefly introduced the latest progress of each quality analyzer, and emphasized the development and application of FTICR and Orbitrap in ntdms analysis.

data processing

除了基本的硬件设施,配套的data processing软件也十分重要。nTDMSdata processing流程通常包括以下4个步骤:同位素峰选取、去卷积、数据库搜索、验证和可视化。正文中,我们对每个步骤进行了简要描述,并重点介绍用于数据库搜索和异质体鉴别的软件。

Acquisition of multi-level structure information

Thanks to the development of various activation/fragmentation methods, ntdms analysis can simultaneously obtain multi-level structural information (Figure 1). There are mainly the following two strategies:the first strategy is that the complete protein complex (MS1) is first broken into subunits (MS2) by CID or SID, and subunits can further fragment peptide segments (MS3). In MS1 and MS2, information such as protein complex binding stoichiometry, topology, protein heterogeneity and so on can be obtained, and in MS3 stage, information such as protein sequence, PTMs localization and heterogeneity source can be obtained. The second strategy is that the intact protein complex (MS1) is directly broken into peptide segments (MS2) by uvpd or Exd, benefiting from the unique fragmentation mode of uvpd and Exd. The region where fragmentation occurs is mainly located in the surface accessible region of the protein complex, while the region where fragmentation does not occur may be located in the core region of the protein complex or participate in the subunit interaction interface. Different fragmentation reflects different spatial structures, and peptide fragments with ligands can be used to locate ligand binding sites. In the review, we described in detail how to use ntdms to obtain multi-level structural information of protein complexes and how to correlate fragment information with structural information.

Figure 1 Multi dimensional structure information provided by ntdms

Applications in complex biological systems

蛋白质的空间结构决定了其生物功能,而蛋白质-蛋白质/配体相互作用是大多数生物进程的基础。通过突变、翻译后修饰、或者与金属、小分子配体、蛋白质、DNA、RNA等分子发生共价或非共价的相互作用,蛋白质功能在活细胞中不断受到调节。随着MS仪器、方法的不断开发和data processing软件的逐渐成熟,nTDMS已被广泛应用于各种生物系统,从小蛋白质、蛋白质-配体复合物到大分子组装体,如Membrane protein、蛋白酶体、核糖体、病毒衣壳,甚至是内源性蛋白混合物。它们中的许多都是极具挑战性的体系,即便是采用NMR、X-射线晶体学或Cryo-EM等生物物理方法分析也是非常困难的。因此,来自nTDMS的见解对于理解这些蛋白质和复合物至关重要。在这里,我们总结nTDMS在所有生物体系中的应用实例,旨在全面了解nTDMS在解决生物学问题方面的潜力。

Structural characterization and differentiation of small proteins

At first, ntdms was mainly used to characterize the structure of monomeric proteins below 50 kDa. Most of the research was carried out around the comparison of gas phase structure and solution phase structure of proteins. According to the fragmentation of ntdms, the gas-phase spatial structure of the protein was deduced and compared with the solution structure obtained by NRM. In addition, if the ion pre activation is added before the secondary fragmentation, it is helpful for the expansion of protein molecules, so as to study the gas phase expansion path of protein and obtain the internal spatial structure information of protein. Thanks to the high sensitivity of fragment ions to the spatial structure of proteins, ntdms is also used to distinguish the structural differences of different protein subtypes and protein mutants.

Protein small molecule ligand interaction

Subsequently, ntdms was applied to protein ligand complexes. Different types of ligands are suitable for different activation/fragmentation methods. Except that electrostatic protein ligands such as metal ions and rna/dna can survive CID activation, most complexes need to choose ECD or uvpd for fragmentation. Ntdms can be used to study protein ligand binding stoichiometry, affinity, binding sites, mechanism of action, structural dynamics/allosteric effects. It is a powerful structural characterization tool, and its application examples in inhibitor screening, enzyme catalytic monitoring, RNA protein interaction mechanism have been introduced in detail in the text.

Protein protein interaction

With the rapid development of instruments and equipment, ntdms has been applied to larger systems, such as protein protein complexes. By combining different activation/fragmentation techniques, multi-level structural information can be obtained in one experiment. Ntdms can help distinguish different protein heterogeneity, and determine the source of heterogeneity at the three levels of complete complex, subunit and peptide. Protein heterogeneity is closely related to its biological function. The transformation of protein function can be achieved by adjusting protein heterogeneity. Specific application cases have been introduced in detail in the text. In addition, ntdms can also be used as protein-protein complex binding interface, gas phase expansion and deep-seated structure exploration.

Therapeutic antibodies and antigen antibody complexes

在过去的几十年中,治疗性抗体已成为最受欢迎的候选药物之一,它们的高特异性和低副作用促进了治疗性抗体的快速增长。在综述中,我们还详细地介绍了nTDMS在Therapeutic antibodies and antigen antibody complexes体系中的应用。nTDMS可用于抗体可变区的测序、具有不同药物计量比(DARs)的抗体耦联药物的结构表征、以及抗体-抗原复合物中互补决定区及抗原表位区的鉴别。

Membrane protein

无论是对于传统的结构表征工具如:X-射线晶体学、NMR还是nTDMS,Membrane protein的结构表征一直以来面临着诸多困难。Membrane protein具有低丰度以及低溶解性等特点,最常见的方法是利用与nMS兼容的膜模拟物如:去污剂胶束、纳米微盘等去溶解Membrane protein,在nTDMS分析时再将Membrane protein从胶束中释放出来,释放出的蛋白可在nTDMS中进一步碎裂获取结构信息。具体的实验流程和应用实例可在综述正文中查看。



Native top-down MS蛋白质组学

值得注意的是,当质谱前端结合非变性分离技术,如native GELFrEE,尺寸排阻色谱,毛细管区带电泳,离子交换色谱等,nTDMS还可以在靶向模式或发现模式下用于复杂蛋白质组的高通量分析,如内源性蛋白混合物。nTDMS分析最大的优势在于它能区分不同的蛋白异质体,并对每种蛋白异质体进行结构表征,这是其他在肽段水平进行分析的结构质谱法如:HDX-MS, CL-MS所无法实现的。





原文:Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes. 



1.Liu RJ, Xia SJ, Li HL. Native top‐down mass spectrometry for higher‐order structural characterization of proteins and complexes. Mass Spec Rev. 2022; e21793.

2.Britt HM, Cragnolini T, Thalassinos K. Integration of mass spectrometry data for structural biology. Chem Rev. 2022;122(8):7952-7986. 

3.Liu XR, Zhang MM, Gross ML. Mass spectrometry-based protein footprinting for higher-order structure analysis:fundamentals and applications. Chem Rev. 2020;120(10):4355-4454.