This article is part of the Mass Spectrometry Applications in Structural Biology special issue. The structure–function relationship is a central theme running throughout biology. As such, study of the molecular structure of biological macromolecules, particularly proteins, nucleic acids, and their complex dynamic interactions, is critical to understanding biological processes. Structural biology has traditionally been dominated by X-ray crystallography and nuclear magnetic resonance spectroscopy. However, a need for complementary biophysical approaches has arisen with the recognition that a combination of structural biology methods is critical to fully characterize details of molecular interactions. Emerging from origins in isotope physics in the early 1900s, and existing primarily in the domain of chemistry until the mid-20th century, dramatic advancements over recent decades have seen mass spectrometry (MS) emerge as a means of studying biological macromolecules. Continued progress has made it possible to transfer delicate biomolecular structures and interactions from solution to the gas phase. These breakthroughs have enabled an ever increasing number of studies applying MS to large biomolecules and their assemblies with increasing analytical depth. Importantly, MS-based approaches typically provide complementary information to traditional biochemical or structural biology methods. The collection of 16 articles in this Thematic Issue highlight some of the recent technological and methodological developments in MS that have been critical to the advancement of biomolecular characterization. They serve to emphasize the unique and powerful insights this technology can provide that underpin a central role for MS in modern structural biology. Key to the role of MS in biology has been the evolution of native mass spectrometry (nMS) in which three-dimensional structures of proteins and their interactors are maintained in the gas phase. nMS has proven invaluable in reporting on higher order structures of proteins and other biomolecules. Heck and co-workers describe how technological innovations in mass analyzers, particularly improving the achievable mass resolving power, have advanced nMS to enable proteoform profiling and structural analysis of complex protein assemblies. This is further expanded by Donald and co-workers with a review focused on applications of nMS in the study of interactions between small molecules and proteins, with implications for drug discovery. nMS is also central to the lofty goal of defining the cellular protein interactome which regulates biological function and dysfunction. In this regard, Rogawski and Sharon describe how nMS can contribute to the study of endogenous protein complexes, purified and studied directly from the host, to retain utmost physiological relevance. Conventional protein MS relies principally on detection of mass to charge (m/z) ratios. In the case of electrospray ionization, most commonly employed to obtain gas-phase biomolecular ions, a distribution of multiply charged ions is typically observed. Meanwhile structural information is often further gleaned by dissociation of intact molecules or assemblies. Traditionally this is achieved by collisions with a neutral gas (collision-induced dissociation), which give rise to characteristic fragment ions that provide insight into the topology and bonding of the assembly. Underlying the continued advancement of MS are key paradigm shifts in the manipulation of ions, from ionization through to fragmentation, separation, and detection. Jarrold provides one such example in his commentary on charge detection MS, a single particle technique enabling accurate mass measurements to be made for heterogeneous biomolecules, frequently not amenable to study by conventional MS. Wysocki and co-workers similarly describe innovative developments in ion activation based on surface collisions to dissociate proteins and protein complexes. This approach, known as surface-induced dissociation, provides powerful information when used in conjunction with complementary structural biology approaches. In parallel with developments in MS instrumentation, significant advances have been made utilizing biological chemistry to modify proteins prior to MS. A comprehensive overview of protein chemistry combined with MS for structure determination is provided by Petrotchenko and Borchers. Together they emphasize the advantages of using various MS experiments to provide constraints for solving protein structures. A number of detailed reviews then follow, describing in depth specific examples of such experiments. Sinz and co-workers summarize reagents and workflows for chemical cross-linking, utilizing prominent examples for characterizing protein 3D structures and protein–protein interactions. Jones and co-workers feature the chemistry of hydroxyl radical footprinting as a means to study the higher order structure of proteins in increasingly complex protein systems. Meanwhile Guttman and co-workers describe how the measurement of hydrogen–deuterium exchange by MS reveals information about not only proteins’ structures but also the dynamics of their diverse conformational states and interactions. Lento and Wilson build on the investigation of protein dynamics through their description of subsecond time-resolved MS approaches. Examples feature the insights gained into the inherently dynamic processes that drive biological function. How these advances in MS and protein chemical labeling are leading to new structural insights directly from cellular environments is reviewed by Bruce and co-workers. In exemplifying cross-linking inside cells in response to various biological processes, they take MS to a new level of cellular structural biology. Linking these approaches together with other MS-based strategies to assess protein stability, Ruotolo and co-workers highlight notable examples where MS-driven protein stability measurements have revealed new insights in biology. Although MS has arguably found greatest recognition in the structural biology arena for its contributions to understanding protein structure, the broad applicability of the technology has led to deep insights regarding the structures of all classes of biomolecules. This is first highlighted in the review by Gabelica and co-workers, who describe the application of structural MS approaches to the study of noncovalent nucleic acid assemblies. Here, they emphasize the ability of MS to unveil new biological roles for specific DNA and RNA structures. Pagel and co-workers also evaluate the novel MS techniques directed at the specific needs of glycan analysis. The complexity of carbohydrates’ structures renders their analysis extremely challenging; however, significant progress in the growing field of glycomics is clearly evident. Beyond the mass spectrometer and data acquisition, the importance of separation and data analysis pipelines in optimizing the information content is becoming increasingly important. Advances in this field have led to an increase in the complexity and heterogeneity of the protein assemblies and interaction networks that can be investigated. Rolland and Prell provide an in-depth review of both theoretical and practical aspects associated with deconvolution of the heterogeneous features of biomolecular assemblies, with particular focus on available algorithms and software tools. Finally, Thalassinos and co-workers review current computational strategies and software solutions to facilitate the integration of diverse MS-based data sets to provide a detailed description of complex biological systems under study. In summary, we expect that this Thematic Issue will serve as an insightful, educational, and enjoyable collection of review articles highlighting the power and exciting future of MS in structural biology. We sincerely thank all the authors for their comprehensive and informative contributions. We are also grateful for the time and dedication of those who participated in the peer review process. Tara Pukala obtained a Ph.D. from the University of Adelaide in 2006, under the supervision of Prof. John Bowie. This was followed by a postdoctoral position at the University of Cambridge, U.K., working with Prof. Dame Carol Robinson in the field of native mass spectrometry. Tara returned to Australia to her current role as lecturer in the Discipline of Chemistry at the University of Adelaide in 2008. Here she leads a multidisciplinary research group focused on developing new approaches, primarily utilizing mass spectrometry and bioconjugation chemistry, to investigate the structure, function, and interactions of macromolecules important in biology and human health. Tara currently serves as President of the Australian and New Zealand society for mass spectrometry and was awarded the 2017 Bowie medal from that society for her contributions to the field. Carol V. Robinson obtained her Ph.D. from the University of Cambridge in 1982, under the supervision of Prof. Dudley Williams. Following a career break of 8-years, she took up a postdoctoral position at the University of Oxford in 1992. She was awarded a Royal Society Research Fellowship in 1995 and returned to Cambridge as Professor in 2000. In 2005 she was awarded a Royal Society Professorship and in 2010 returned to Oxford as the Dr. Lees Professor of Physical Chemistry, a position she holds to this day. In 2021 she became the inaugural director of the Kavli Institute for Nanoscience Discovery in Oxford, an interdisciplinary science centre comprised of eight science departments with remit to bring the physical sciences into the cell. This article has not yet been cited by other publications.

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简介：质谱在结构生物学中的应用

本文是部分质谱在结构生物学中的应用特刊。结构-功能关系是贯穿整个生物学的中心主题。因此，研究生物大分子的分子结构，特别是蛋白质、核酸及其复杂的动态相互作用，对于理解生物过程至关重要。结构生物学传统上以 X 射线晶体学和核磁共振光谱学为主。然而，随着人们认识到结构生物学方法的组合对于充分表征分子相互作用的细节至关重要，因此需要补充生物物理方法。起源于 1900 年代早期的同位素物理学，主要存在于化学领域，直到 20 世纪中叶，近几十年来的巨大进步使质谱 (MS) 成为研究生物大分子的一种手段。持续的进步使得将精细的生物分子结构和相互作用从溶液转移到气相成为可能。这些突破使得越来越多的研究将 MS 应用于大型生物分子及其组装体，并增加了分析深度。重要的是，基于 MS 的方法通常为传统的生化或结构生物学方法提供补充信息。本专题收录了 16 篇文章，重点介绍了 MS 近期的一些技术和方法发展，这些发展对生物分子表征的进步至关重要。它们有助于强调这项技术可以提供的独特而强大的洞察力，这些洞察力支撑了 MS 在现代结构生物学中的核心作用。MS 在生物学中的作用的关键是天然质谱 (nMS) 的发展，其中蛋白质及其相互作用物的三维结构保持在气相中。nMS 已被证明在报告蛋白质和其他生物分子的高阶结构方面非常宝贵。Heck 和他的同事描述了质量分析仪的技术创新，特别是提高了可实现的质量分辨能力，如何拥有先进的 nMS 以实现复杂蛋白质组装的蛋白质形式分析和结构分析。Donald 和他的同事进一步扩展了这一点，并回顾了 nMS 在小分子和蛋白质相互作用研究中的应用，对药物发现有影响。nMS 也是定义调节生物功能和功能障碍的细胞蛋白质相互作用组的崇高目标的核心。在这方面，Rogawski 和 Sharon 描述了 nMS 如何有助于直接从宿主中纯化和研究的内源性蛋白质复合物的研究，以保持最大的生理相关性。传统的蛋白质质谱主要依赖于质量与电荷的检测（米/ z) 比率。在电喷雾电离的情况下，最常用于获得气相生物分子离子，通常观察到多电荷离子的分布。同时，结构信息通常通过完整分子或组件的解离进一步收集。传统上，这是通过与中性气体的碰撞（碰撞诱导解离）来实现的，这会产生特征碎片离子，从而深入了解组件的拓扑结构和键合。MS 不断进步的基础是离子操作的关键范式转变，从电离到碎裂、分离和检测。Jarrold 在他对电荷检测 MS 的评论中提供了一个这样的例子，这是一种单粒子技术，可以对异质生物分子进行精确的质量测量，通常不适合通过传统 MS 进行研究。Wysocki 及其同事类似地描述了基于表面碰撞解离蛋白质和蛋白质复合物的离子活化的创新发展。这种被称为表面诱导解离的方法在与互补的结构生物学方法结合使用时提供了强大的信息。在 MS 仪器发展的同时，利用生物化学在 MS 之前修饰蛋白质也取得了重大进展。Petrotchenko 和 Borchers 提供了蛋白质化学与 MS 结合用于结构测定的全面概述。他们一起强调了使用各种 MS 实验为解决蛋白质结构提供约束的优势。然后是一些详细的评论，深入描述此类实验的具体示例。Sinz 及其同事总结了化学交联的试剂和工作流程，利用突出的例子来表征蛋白质 3D 结构和蛋白质-蛋白质相互作用。Jones 及其同事将羟基自由基足迹化学作为研究日益复杂的蛋白质系统中蛋白质高级结构的一种手段。与此同时，Guttman 及其同事描述了 MS 对氢-氘交换的测量如何不仅揭示了蛋白质结构的信息，而且揭示了它们不同构象状态和相互作用的动力学。Lento 和 Wilson 通过描述亚秒级时间分辨 MS 方法来研究蛋白质动力学。示例展示了对驱动生物功能的内在动态过程的见解。Bruce 及其同事回顾了 MS 和蛋白质化学标记的这些进展如何直接从细胞环境中获得新的结构见解。在举例说明细胞内响应各种生物过程的交联时，他们将 MS 带到了细胞结构生物学的新水平。将这些方法与其他基于 MS 的策略联系起来以评估蛋白质稳定性，Ruotolo 和他的同事强调了一些值得注意的例子，其中 MS 驱动的蛋白质稳定性测量揭示了生物学的新见解。尽管 MS 可以说因其对理解蛋白质结构的贡献而在结构生物学领域获得了最大的认可，该技术的广泛适用性使人们对所有类别的生物分子的结构有了深入的了解。Gabelica 及其同事在评论中首先强调了这一点，他们描述了结构 MS 方法在非共价核酸组装研究中的应用。在这里，他们强调 MS 能够揭示特定 DNA 和 RNA 结构的新生物学作用。Pagel 及其同事还评估了针对聚糖分析特定需求的新型 MS 技术。碳水化合物结构的复杂性使其分析极具挑战性；然而，在不断发展的糖组学领​​域取得的重大进展是显而易见的。除了质谱仪和数据采集，分离和数据分析管道在优化信息内容方面的重要性变得越来越重要。该领域的进步导致可以研究的蛋白质组装和相互作用网络的复杂性和异质性增加。Rolland 和 Prell 对与生物分子组装的异构特征的反卷积相关的理论和实践方面进行了深入的回顾，特别关注可用的算法和软件工具。最后，Thalassinos 及其同事回顾了当前的计算策略和软件解决方案，以促进各种基于 MS 的数据集的集成，以提供对正在研究的复杂生物系统的详细描述。总之，我们希望本专题将成为一个富有洞察力的、具有教育意义的、和令人愉快的评论文章集，突出了 MS 在结构生物学中的力量和令人兴奋的未来。我们衷心感谢所有作者的全面而翔实的贡献。我们也感谢那些参与同行评审过程的人所付出的时间和奉献精神。Tara Pukala 获得博士学位。2006 年毕业于阿德莱德大学，师从 John Bowie 教授。随后在英国剑桥大学担任博士后职位，与 Dame Carol Robinson 教授在原生质谱领域合作。塔拉于 2008 年回到澳大利亚，担任阿德莱德大学化学学科的讲师。在这里，她领导了一个专注于开发新方法的多学科研究小组，主要利用质谱和生物共轭化学，研究对生物学和人类健康很重要的大分子的结构、功能和相互作用。Tara 目前担任澳大利亚和新西兰质谱学会主席，并因其对该领域的贡献而获得该学会颁发的 2017 年鲍伊奖章。Carol V. Robinson 获得了博士学位。1982 年从剑桥大学毕业，师从 Dudley Williams 教授。在中断了 8 年的职业生涯后，她于 1992 年在牛津大学担任博士后职位。她于 1995 年获得皇家学会研究奖学金，并于 2000 年返回剑桥担任教授。2005 年，她获得了皇家学会授予教授职位，并于 2010 年回到牛津，担任 Lees 博士物理化学教授，她一直担任这一职务。2021 年，她成为牛津大学 Kavli 纳米科学研究所的首任主任，这是一个由八个科学部门组成的跨学科科学中心，负责将物理科学带入细胞。这篇文章尚未被其他出版物引用。