Mass spectrometry for structural characterization of therapeutic antibodies

Antibodies, also known as immunoglobulins, have emerged as one of the most promising classes of therapeutics in the biopharmaceutical industry. The need for complete characterization of the quality attributes of these molecules requires sophisticated techniques. Mass spectrometry (MS) has become an essential analytical tool for the structural characterization of therapeutic antibodies, due to its superior resolution over other analytical techniques. It has been widely used in virtually all phases of antibody development. Structural features determined by MS include amino acid sequence, disulfide linkages, carbohydrate structure and profile, and many different post-translational, in-process, and in-storage modifications. In this review, we will discuss various MS-based techniques for the structural characterization of monoclonal antibodies. These techniques are categorized as mass determination of intact antibodies, and as middle-up, bottom-up, top-down, and middle-down structural characterizations. Each of these techniques has its advantages and disadvantages in terms of structural resolution, sequence coverage, sample consumption, and effort required for analyses. The role of MS in glycan structural characterization and profiling will also be discussed.     

Fourier-transform ion cyclotron resonance mass spectrometry for characterizing proteoforms

Proteoforms contribute functional diversity to the proteome and aberrant proteoforms levels have been implicated in biological dysfunction and disease. Fourier-transform ion cyclotron resonance mass spectrometry (FT-ICR MS), with its ultrahigh mass-resolving power, mass accuracy, and versatile tandem MS capabilities, has empowered top-down, middle-down, and native MS-based approaches for characterizing proteoforms and their complexes in biological systems. Herein, we review the features which make FT-ICR MS uniquely suited for measuring proteoform mass with ultrahigh resolution and mass accuracy; obtaining in-depth proteoform sequence coverage with expansive tandem MS capabilities; and unambiguously identifying and localizing post-translational and noncovalent modifications. We highlight examples from our body of work in which we have quantified and comprehensively characterized proteoforms from cardiac and skeletal muscle to better understand conditions such as chronic heart failure, acute myocardial infarction, and sarcopenia. Structural characterization of monoclonal antibodies and their proteoforms by FT-ICR MS and emerging applications, such as native top-down FT-ICR MS and high-throughput top-down FT-ICR MS-based proteomics at 21 T, are also covered. Historically, the information gleaned from FT-ICR MS analyses have helped provide biological insights. We predict FT-ICR MS will continue to enable the study of proteoforms of increasing size from increasingly complex endogenous mixtures and facilitate the benchmarking of sensitive and specific assays for clinical diagnostics. © 2020 John Wiley & Sons Ltd. Mass Spec Rev     

应用基于非变性质谱及串联质谱的碎片互补法分析完整IgA2抗体及其尺寸变异体

在完整蛋白及其复合物层面表征糖蛋白可提供修饰程度、变异体分布、结合计量关系等方面的重要结构信息。大量天然蛋白质及蛋白质药物都以糖蛋白形式存在,随着糖基化程度的增大,蛋白体系的异质性增强,糖链与肽链理化性质的差异使得常规表征手段容易损失准确性。使用质谱测定会因蛋白型质量分布展宽造成信号重叠,以及不同糖型离子化响应差异造成不同价态蛋白型质量分布差异而损失准确度,甚至无法实现分子质量测定。为解决这一问题,开发了基于有限价态还原的非变性质谱方法,但这些方法有的存在还原效率有限,有的需使用仅个别型号商品化仪器才具备的基于电子/质子转移的气相反应功能,应用范围受到限制。本工作针对含有非共价亚基的完整IgA2单克隆抗体及其尺寸变异体复合物体系,使用基于非变性质谱及串联质谱的碎片互补方法,以及碰撞诱导解离(CID)或高能碰撞解离(HCD)反应,综合利用有限价态还原效应和解离反应的质量守恒约束条件,实现高异质性糖蛋白复合物的测定。通过与针对全体蛋白型系综还原方法及蛋白型亚群有限还原方法的平行比较和交叉验证,评价各种方法的性能。结果表明,在解离产物特异性高且分子质量均一的情况下,碎片互补法可充分保证价态及...     

Detection and localization of protein modifications by high resolution tandem mass spectrometry

For interrogation of peptides with diverse modifications, no other instrument is as versatile as the Fourier-transform mass spectrometer (FTMS). Particularly using electrospray ionization (ESI), many intact proteins and their proteolytic products harboring post-translational and chemical modifications (PTMs) have been studied by high resolution tandem mass spectrometry (MS/MS). The widely touted analytical figures of merit for FTMS in fact have translated into clarity when analyzing PTMs from phosphorylations to disulfides, oxidations, methylations, acetylations, and even exotic PTMs found in the biosynthesis of antibiotics and other natural products. A top down approach to PTM detection and localization is proving extensible to an increasing variety of PTMs, some of which are stable to MS/MS at the protein level but unstable to amide bond cleavage by threshold dissociations at the level of small peptides <3 kDa. In contrast, MS/MS using electron capture dissociation (ECD) allows precise localization of even labile PTMs given enough sample and abundant molecular ions. Finally, this brief synopsis of recent literature highlights specific PTMs that perturb the protein backbone therefore altering MS/MS fragmentation patterns. Thus, FTMS will continue its expansion into more laboratories in part because of its ability to detect and deconvolute the regulatory mechanisms of biology written in the language of PTMs.     

Protein sequence information by matrix-assisted laser desorption/ionization in-source decay mass spectrometry

Proteins from biological samples are often identified by mass spectrometry (MS) with the two following "bottom-up" approaches: peptide mass fingerprinting or peptide sequence tag. Nevertheless, these strategies are time-consuming (digestion, liquid chromatography step, desalting step), the N- (or C-) terminal information often lacks and post-translational modifications (PTMs) are hardly observed. The in-source decay (ISD) occurring in a matrix assisted laser desorption/ionization (MALDI) source appears an interesting analytical tool to obtain N-terminal sequence, to identify proteins and to characterize PTMs by a "top-down" strategy. The goal of this review deals with the usefulness of the ISD technique in MALDI source in proteomics fields. In the first part, the ISD principle is explained and in the second part, the use of ISD in proteomic studies is discussed for protein identification and sequence characterization.     

Discovery of Novel Biochemical Pathways Using Proteomics Approaches

Extensive studies in histones and p53 suggest that these proteins can be modified by an array of protein post-translational modifications (PTMs), most of which are correlated with important biological functions. Nevertheless, PTM types and their sites in other proteins remain largely unknown, except several widely studied ones. The information deficiency suggests an urgent need for reliable technology for characterization of all the PTMs in a protein and for their dynamic studies. To this end, we recently developed an unrestrictive protein sequence alignment algorithm, PTMap, and used it in conjunction with mass spectrometry for accurate identification of all the possible PTMs, known or undescribed ones. We have used the algorithm to identify novel PTMs, to discover common in vitro protein modifications, to study PTM cross-talks, and to study protein mutations. In this presentation, applications of such mass spectrometry-based proteomics technologies will be discussed.     

Microproteomics: analysis of protein diversity in small samples

Proteomics, the large-scale study of protein expression in organisms, offers the potential to evaluate global changes in protein expression and their post-translational modifications that take place in response to normal or pathological stimuli. One challenge has been the requirement for substantial amounts of tissue in order to perform comprehensive proteomic characterization. In heterogeneous tissues, such as brain, this has limited the application of proteomic methodologies. Efforts to adapt standard methods of tissue sampling, protein extraction, arraying, and identification are reviewed, with an emphasis on those appropriate to smaller samples ranging in size from several microliters down to single cells. The effects of miniaturization on these analyses are highlighted using neuroscience-related examples, as are statistical issues unique to the high-dimensional datasets generated by proteomic experiments.     

Discovering new invertebrate neuropeptides using mass spectrometry

Neuropeptides are a complex set of messenger molecules controlling a wide array of regulatory functions and behaviors within an organism. These neuromodulators are cleaved from longer protein molecules and often experience numerous post-translational modifications to achieve their bioactive form. As a result of this complexity, sensitive and versatile analysis schemes are needed to characterize neuropeptides. Mass spectrometry (MS) through a variety of approaches has fueled the discovery of hundreds of neuropeptides in invertebrate species in the last decade. Particularly successful are direct tissue and single neuron analyses by matrix-assisted laser desorption/ionization (MALDI) MS, which has been used to elucidate approximately 440 neuropeptides, and examination of neuronal homogenates by electrospray ionization techniques (ESI), also leading to the characterization of over 450 peptides. Additional MS methods with great promise for the discovery of neuropeptides are MS imaging and large-scale peptidomics studies in combination with a sequenced genome.     

A systematic approach to the discovery of new proteins of ultrastructural interest

A systematic approach to the discovery of new proteins of ultrastructural interest is discussed. It involves the merging of monoclonal antibody technology with immunocytochemical technology, particularly immunoelectron microscopy. In this approach, monoclonal antibodies are raised to a cellular preparation that can be grossly heterogeneous in its protein composition. The hybridoma culture fluids are screened by immunocytochemistry for the ultrastructural localization of their antibodies. Those monoclonal antibodies that show specific ultrastructural localizations of interest are then selected for further investigation. The antigen to which a given monoclonal antibody is directed is then identified by immunoprecipitation and immunoblotting with that antibody. By this approach, two new striated muscle proteins of ultrastructural interest have been discovered and are named zeugmatin and enactin. The former is a protein of over 500 kD localized by immunoelectron microscopy to the Z-bands, the latter of 245 kD localized to the N₁ line of striated muscle.     

Informatics for mass spectrometry-based RNA analysis

Mass spectrometry (MS) allows the sensitive and direct characterization of biological macromolecules and therefore has the potential to complement the more conventional genetic and biochemical methods used for RNA characterization. Although MS has been used much less frequently for RNA research than it has been for protein research, recent technical improvements in both instrumentation and software make MS a powerful tool for RNA analysis because it can now be used to sequence, quantify, and chemically analyze RNAs. Mass spectrometry is particularly well suited for the characterization of RNAs associated with ribonucleoprotein complexes. This review focuses on the software and databases that can be used for MS-based RNA studies. Software for the processing of raw mass spectra, the identification and characterization of RNAs by mass mapping, de novo sequencing, and tandem MS-based database searching are available.     

非变性结构质谱在蛋白提取、离子源开发与构象解析方面的研究进展

非变性条件下的蛋白质研究可以提供更准确的高级结构信息,为“序列-结构-功能”关系解析提供更接近生理条件的分子基础。非变性质谱(native MS)是在非变性条件下对目标蛋白质进行质谱分析的技术。近年来,非变性质谱在蛋白构象、蛋白质非共价相互作用以及蛋白质组装复合物等高阶结构解析中发挥着不可替代的作用,并逐渐成为结构生物学研究的重要支撑方法之一。本文总结了非变性结构质谱在蛋白提取、离子源开发与构象解析方面的发展现状,并提出了非变性结构质谱领域现阶段的技术瓶颈与潜在的解决策略,主要包含原位分析能力和结构分辨率的提升这两方面。     

Electrospray ionization mass spectrometry of oligonucleotide complexes with drugs, metals, and proteins.

I. Introduction 61 II. Binding of Small Molecules to DNA 62 A. Covalent Binding 62 B. Reversible (Noncovalent) DNA-Binding Agents 65 III. DNA-Metal Ion Complexes 67 A. Platinum Complexes 70 B. Other Metal Ions 73 IV. DNA-Protein Complexes 74 A. Introduction 74 B. ESI-MS of DNA-Protein Complexes 76 C. ESI-MS Analysis of Proteolytic Products of DNA-Protein Complexes 79 D. ESI-MS of Ternary DNA-Protein-Ligand Complexes 80 V. Conclusions 80 Abbreviations 81 References 81 --Interactions of DNA with drugs, metal ions, and proteins are important in a wide variety of biological processes. With the advent of electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI), mass spectrometry (MS) is now a well-established tool for the characterization of the primary structures of biopolymers. The gentle nature of the ESI process, however, means that ESI-MS is also finding application for the study of noncovalent and other fragile biomolecular complexes. We outline here the progress, to date, in the use of ESI-MS for the study of noncovalent drug-DNA and protein-DNA complexes together with strategies that can be employed to examine the binding of small molecules and metal complexes to DNA. In the case of covalent complexes with DNA, sequence information can be derived from ESI-MS used in conjunction with tandem mass spectrometry (MS/MS) and/or enzymatic digestion. MS/MS can also be used to probe the relative binding affinities of drugs that bind to DNA via noncovalent interactions. Overall, the work in this area, to date has demonstrated that ESI-MS and MS/MS will prove to be valuable complements to other structural methods, offering advantages in terms of speed, specificity, and sensitivity. (c) 2001 John Wiley & Sons, Inc.     

ApplicationofMassSpectrometryinPharmaceuticalDrugDiscoveryResearch

Mass spectrometry has been extensively utilized in drug discovery research and development, from the traditional medicinal chemistry support to drug metabolism and pharmacokinetic studies. As the industry faces with the challenges of tougher diseases and more challenging pathways/targets to tackle, it requires the development of innovative solutions to address needs from target discovery to biomarker identification. It also presents a great opportunity for scientists in the field for innovation, many of which often come from cross disciplinary efforts - chemistry, biology and technology. In this presentation, four areas of drug discovery research where innovative solutions have been developed will be presented: (1) target identification and validation through cellular metabolomic analysis and chemical proteomics; (2) hits discovery and validation using MS-based screening; (3) PK analysis of therapeutic monoclonal antibody; (4) MS imaging for drug tissue distribution. Investment in life saving and life style improving medicines will get more intensified as standard of living improves. Drug discovery research will continue to require and present opportunity for such application driven innovations.     

Prospective highlights of functional skin proteomics

Although a wide variety of protein profiles have been extensively constructed via proteomic analysis, the comprehensive proteomic profiling of the skin, which is considered to be the largest organ of the human body, is still far from complete. Our efforts to establish the functional skin proteome, a protein database describing the protein networks that underlie biological processes, has set in motion the identification and characterization of proteins expressed in the epidermis and dermis of the BALB/c mice. In this review, we will highlight various cutaneous proteins we have characterized and discuss their biological functions associated with skin distress, immunity, and cancer. This type of research into functional skin proteomics will provide a critical step toward understanding disease and developing successful therapeutic strategies.     

Synchronization of heterogeneous linear networks with distinct inner coupling matrices

In this paper, we study synchronization of heterogeneous linear networks with distinct inner coupling matrices. Firstly, for synchronous networks, we show that any synchronous trajectory will converge to a corresponding synchronous state. Then, we provide an invariant set, which can be exactly obtained by solving linear equations and then used for characterizing synchronous states. Afterwards, we use inner coupling matrices and node dynamics to successively decompose the original network into a new network, composed of the external part and the internal part. Moreover, this new network can be proved to synchronize to the above invariant set by constructing the corresponding desired Lyapunov-like functions for the internal part and the external part respectively. In particular, this result still holds if the coupling strength is disturbed slightly. Finally, examples with numerical simulations are given to illustrate the validity and applicability of our theoretical results.     

Top‐down mass spectrometry for the analysis of combinatorial post‐translational modifications

Protein post‐translational modifications (PTMs) are critically important in regulating both protein structure and function, often in a rapid and reversible manner. Due to its sensitivity and vast applicability, mass spectrometry (MS) has become the technique of choice for analyzing PTMs. Whilst the “bottom‐up' analytical approach, in which proteins are proteolyzed generating peptides for analysis by MS, is routinely applied and offers some advantages in terms of ease of analysis and lower limit of detection, “top‐down” MS, describing the analysis of intact proteins, yields unique and highly valuable information on the connectivity and therefore combinatorial effect of multiple PTMs in the same polypeptide chain. In this review, the state of the art in top‐down MS will be discussed, covering the main instrumental platforms and ion activation techniques. Moreover, the way that this approach can be used to gain insights on the combinatorial effect of multiple post‐translational modifications and how this information can assist in studying physiologically relevant systems at the molecular level will also be addressed. © 2012 Wiley Periodicals, Inc., Mass Spec Rev 32:27–42, 2013     

Improved Performance of a Tandem Quadrupole/Time-of-Flight Mass Spectrometer

Abstract

This paper describes the use of a time-of-flight mass spectrometer as the second stage of a MS/MS instrument. This system has been improved by the addition of a newly designed data acquisition system. The performance of this instrument will be discussed along with other modifications that are in progress to give further improvements.