Technologies for PTM discovery and functional mapping p. 505

项目摘要 蛋白质的翻译后修饰（PTM）是细胞信号传导的一种普遍形式，其协调许多细胞信号传导。 过程，包括代谢、细胞移动性、细胞周期和分化。因此，不适当的监管 PTMs广泛涉及异常发育和疾病。翻译后修饰的机制 提供了一种快速且大部分可逆的方法来调节蛋白质活性和信号转导。因此 蛋白质组及其修饰代表了丰富和信息丰富的实验平台。研究旨在 了解它的动力学无疑将促进我们对基础生物学和疾病的理解。质量 质谱法（MS）非常适合于蛋白质组学分析，因为它具有高度灵敏度， 与基于抗体的方法不同，该方法不需要蛋白质的先验知识， 靶点或翻译后修饰;事实上，MS已用于对各种翻译后修饰的复杂性进行编目 非常详细。尽管如此，仍然存在两大挑战。首先，蛋白质组学领域几乎一致地 依赖于肽阳离子分析（即，正电喷雾）。这种形式的结果是酸性PTM， 以及蛋白质组酸性区域中包含的所有PTM，通常很难或不可能检测到， 它们在标准的低pH条件下不能有效地溶解。我们通过发展 高pH分离方法沿着负电子转移解离（NETD）。NETD允许 肽阴离子的测序，并应允许访问以前未观察到的PTM和部分的 蛋白质组第二个主要挑战是如何将大型PTM数据集转换为生物信息。到 针对这个问题，我们提出了一种高通量技术，酶特异性的多重测定（MAES）， 其通过内切酶底物-酶作图将发现PTM数据转化为功能信息。映射 负责特定修饰事件的酶是有指导意义的，因为它将那些修饰置于 指导生物功能的信号分子的背景。中心的许多DBP将建立 全面的PTM列表，这将作为进一步研究的基础。为这些提供方向 研究，有针对性的，但可扩展的，测定是必要的，以映射酶的调节修饰位点， 兴趣

Project Summary Post-translational modification (PTM) of proteins is a pervasive form of cell signaling that orchestrates numerous processes, including metabolism, cell mobility, cell cycle, and differentiation. As a result, improper regulation of PTMs is widely implicated in aberrant development and disease. Mechanistically, post-translational modification provides a rapid and largely reversible means to modulate protein activity and transduce signals. Thus, the proteome and its modifications represent a rich and informative experimental plane. Research that seeks to understand its dynamics will doubtless advance our understanding of fundamental biology and disease. Mass spectrometry (MS) is well-suited to proteomic analysis because it is highly sensitive, has the capacity to localize PTMs to a single amino acid, and, unlike antibody-based methods, does not require a priori knowledge of protein targets or post-translational modifications; indeed, MS has been used to catalog the complexity of various PTMs with great detail. That said, two major challenges remain. First, the field of proteomics almost uniformly relies on peptide cation analysis (i.e., positive electrospray). The consequence of this format is that acidic PTMs, and all PTMs contained in acidic regions of the proteome, are often difficult, or impossible, to detect because they do not ionize effectively under the standard low-pH conditions. We confront this problem by developing high-pH separations methodology along with negative electron transfer dissociation (NETD). NETD allows for the sequencing of peptide anions and should permit access to previously unobserved PTMs and portions of the proteome. The second major challenge is how to convert large PTM datasets to biological information. To counter this issue we propose a high-throughput technology, multiplexed assay for enzyme specificity (MAES), that converts discovery PTM data to functional information by en masse substrate-to-enzyme mapping. Mapping the enzymes responsible for a specific modification event is instructive because it places those modifications in the context of signaling molecules that direct biological function. Many of the DBPs in the Center will establish comprehensive lists of PTMs, which will serve as the foundation for further study. To provide direction for these studies, targeted, yet scalable, assays are necessary to map the enzymes that regulate modification sites of interest.