Zhou Shufeng
Department of Pharmacy, Faculty of Science, National University of Singapore, Science Drive 4, Singapore 117543, Singapore.
J Chromatogr B Analyt Technol Biomed Life Sci. 2003 Nov 25;797(1-2):63-90. doi: 10.1016/s1570-0232(03)00399-4.
Covalent binding of reactive metabolites of drugs to proteins has been a predominant hypothesis for the mechanism of toxicity caused by numerous drugs. The development of efficient and sensitive analytical methods for the separation, identification, quantification of drug-protein adducts have important clinical and toxicological implications. In the last few decades, continuous progress in analytical methodology has been achieved with substantial increase in the number of new, more specific and more sensitive methods for drug-protein adducts. The methods used for drug-protein adduct studies include those for separation and for subsequent detection and identification. Various chromatographic (e.g., affinity chromatography, ion-exchange chromatography, and high-performance liquid chromatography) and electrophoretic techniques [e.g., sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional SDS-PAGE, and capillary electrophoresis], used alone or in combination, offer an opportunity to purify proteins adducted by reactive drug metabolites. Conventionally, mass spectrometric (MS), nuclear magnetic resonance, and immunological and radioisotope methods are used to detect and identify protein targets for reactive drug metabolites. However, these methods are labor-intensive, and have provided very limited sequence information on the target proteins adducted, and thus the identities of the protein targets are usually unknown. Moreover, the antibody-based methods are limited by the availability, quality, and specificity of antibodies to protein adducts, which greatly hindered the identification of specific protein targets of drugs and their clinical applications. Recently, the use of powerful MS technologies (e.g., matrix-assisted laser desorption/ionization time-of-flight) together with analytical proteomics have enabled one to separate, identify unknown protein adducts, and establish the sequence context of specific adducts by offering the opportunity to search for adducts in proteomes containing a large number of proteins with protein adducts and unmodified proteins. The present review highlights the separation and detection technologies for drug-protein adducts, with an emphasis on methodology, advantages and limitations to these techniques. Furthermore, a brief discussion of the application of these techniques to individual drugs and their target proteins will be outlined.
药物活性代谢产物与蛋白质的共价结合一直是众多药物所致毒性机制的主要假说。开发高效、灵敏的分析方法用于药物 - 蛋白质加合物的分离、鉴定和定量具有重要的临床和毒理学意义。在过去几十年中，分析方法不断取得进展，用于药物 - 蛋白质加合物的新的、更特异、更灵敏的方法数量大幅增加。用于药物 - 蛋白质加合物研究的方法包括分离方法以及后续的检测和鉴定方法。各种色谱技术（如亲和色谱、离子交换色谱和高效液相色谱）和电泳技术[如十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳（SDS - PAGE）、二维SDS - PAGE和毛细管电泳]，单独使用或联合使用，为纯化被活性药物代谢产物加合的蛋白质提供了机会。传统上，质谱（MS）、核磁共振以及免疫和放射性同位素方法用于检测和鉴定活性药物代谢产物的蛋白质靶点。然而，这些方法 labor-intensive，并且提供的关于被加合靶点蛋白质的序列信息非常有限，因此蛋白质靶点的身份通常未知。此外，基于抗体的方法受到针对蛋白质加合物抗体的可用性、质量和特异性的限制，这极大地阻碍了药物特异性蛋白质靶点的鉴定及其临床应用。最近，强大的MS技术（如基质辅助激光解吸/电离飞行时间）与分析蛋白质组学的结合使用，使得人们能够分离、鉴定未知的蛋白质加合物，并通过在包含大量有蛋白质加合物和未修饰蛋白质的蛋白质组中搜索加合物，建立特定加合物的序列背景。本综述重点介绍了药物 - 蛋白质加合物的分离和检测技术，重点阐述了这些技术的方法、优点和局限性。此外，还将简要讨论这些技术在个别药物及其靶点蛋白质上的应用。
