Proteins physiologically exist as "proteoforms" that arise from one gene and acquire additional function by post-translational modifications (PTM). When multiple PTMs coexist on single protein molecules, top-down proteomics becomes the only feasible method of characterization; however, most top-down methods have limited quantitative capacity and insufficient throughput to truly address proteoform biology. Here we demonstrate that top-down proteomics can be quantitative, reproducible, sensitive, and high throughput. The proteoforms of histone H4 are well studied both as a challenging proteoform identification problem and due to their essential role in the regulation of all eukaryotic DNA-templated processes. Much of histone H4's function is obfuscated from prevailing methods due to combinatorial mechanisms. Starting from cells or tissues, after an optimized protein purification process, the H4 proteoforms are physically separated by on-line C3 chromatography, narrowly isolated in MS1 and sequenced with ETD fragmentation. We achieve more than 30 replicates from a single 35-mm tissue culture dish by loading 55 ng of H4 on column. Parallelization and automation yield a sustained throughput of 12 replicates per day. We achieve reproducible quantitation (average biological Pearson correlations of 0.89) of hundreds of proteoforms (about 200-300) over almost six orders of magnitude and an estimated LLoQ of 0.001% abundance. We demonstrate the capacity of the method to precisely measure well-established changes with sodium butyrate treatment of SUM159 cells. We show that the data produced by a quantitative top-down method can be amenable to parametric statistical comparisons and is capable of delineating relevant biological changes at the full proteoform level.

中文翻译：

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高通量定量自上而下蛋白质组学：组蛋白 H4。

蛋白质在生理上以“蛋白质型”的形式存在，它由一个基因产生并通过翻译后修饰 (PTM) 获得额外的功能。当多个 PTM 共存于单个蛋白质分子时，自上而下的蛋白质组学成为唯一可行的表征方法；然而，大多数自上而下的方法定量能力有限，吞吐量不足，无法真正解决蛋白质形态生物学问题。在这里，我们证明自上而下的蛋白质组学可以是定量的、可重复的、灵敏的和高通量的。组蛋白 H4 的蛋白质型作为具有挑战性的蛋白质型鉴定问题以及由于它们在调节所有真核 DNA 模板化过程中的重要作用而得到了很好的研究。由于组合机制，组蛋白 H4 的大部分功能与流行方法混淆。从细胞或组织开始，经过优化的蛋白质纯化过程后，H4 蛋白型通过在线 C3 层析进行物理分离，在 MS1 中进行严格分离，并使用 ETD 片段进行测序。通过在柱上加载 55 ng H4，我们从单个 35 毫米组织培养皿中获得了 30 多个重复。并行化和自动化产生每天 12 次重复的持续吞吐量。我们实现了近六个数量级的数百种蛋白质型（约 200-300）的可重复定量（平均生物 Pearson 相关系数为 0.89），估计 LLoQ 为 0.001% 丰度。我们证明了该方法能够精确测量丁酸钠处理 SUM159 细胞时已确定的变化。