Top-down proteomics is emerging as a preferred approach to investigate biological systems, with objectives ranging from the detailed assessment of a single protein therapeutic, to the complete characterization of every possible protein including their modifications, which define the human proteoform. Given the controlling influence of protein modifications on their biological function, understanding how gene products manifest or respond to disease is most precisely achieved by characterization at the intact protein level. Top-down mass spectrometry (MS) analysis of proteins entails unique challenges associated with processing whole proteins while maintaining their integrity throughout the processes of extraction, enrichment, purification, and fractionation. Recent advances in each of these critical front-end preparation processes, including minimalistic workflows, have greatly expanded the capacity of MS for top-down proteome analysis. Acknowledging the many contributions in MS technology and sample processing, the present review aims to highlight the diverse strategies that have forged a pathway for top-down proteomics. We comprehensively discuss the evolution of front-end workflows that today facilitate optimal characterization of proteoform-driven biology, including a brief description of the clinical applications that have motivated these impactful contributions.

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在 MS 分析之前自上而下的蛋白质组样本处理的最新进展

自上而下的蛋白质组学正在成为研究生物系统的首选方法，其目标范围从对单一蛋白质治疗的详细评估到对每种可能的蛋白质的完整表征，包括它们的修饰，这些修饰定义了人类蛋白质组。鉴于蛋白质修饰对其生物学功能的控制影响，了解基因产物如何表现或对疾病作出反应最精确地通过完整蛋白质水平的表征来实现。蛋白质的自上而下质谱 (MS) 分析带来了与处理整个蛋白质相关的独特挑战，同时在整个提取、富集、纯化和分馏过程中保持其完整性。这些关键的前端准备过程中的每一个的最新进展，包括简约的工作流程，极大地扩展了 MS 进行自上而下的蛋白质组分析的能力。承认 MS 技术和样品处理的许多贡献，本综述旨在强调为自上而下的蛋白质组学打造途径的多种策略。我们全面讨论了前端工作流程的演变，这些工作流程今天促进了蛋白质驱动生物学的最佳表征，包括对激发这些有影响力贡献的临床应用的简要描述。