Technologies for PTM discovery and functional mapping p. 505

项目摘要 蛋白质的翻译后修饰(PTM)是一种普遍存在的细胞信号形式，它协调了许多 过程，包括新陈代谢、细胞移动、细胞周期和分化。因此，不适当的监管 PTMS被广泛地与异常发育和疾病有关。从机械上讲，翻译后修饰 提供了一种快速且在很大程度上可逆的方法来调节蛋白质的活性和转导信号。因此， 蛋白质组及其修饰代表了一个丰富和信息丰富的实验平面。这项研究试图 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 光谱分析非常适合蛋白质组分析，因为它具有高度的灵敏度，具有定位的能力 与基于抗体的方法不同，PTMS不需要蛋白质的先验知识 目标或翻译后修改；实际上，已使用MS来分类各种PTM的复杂性 非常详细。话虽如此，但仍存在两大挑战。首先，蛋白质组学领域几乎一致 依靠多肽阳离子分析(即正电喷雾)。这种形式的结果是酸性的PTMS， 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，因为 它们在标准的低pH值条件下不能有效地电离。面对这一问题，我们通过发展 高pH分离方法和负电子转移解离(NETD)。NETD允许 多肽阴离子的测序，并应允许访问以前未观察到的PTM和部分 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。至 针对这一问题，我们提出了一种高通量技术，即酶特异性(MAES)的多重检测， 它通过整体底物到酶的映射将发现的PTM数据转换为功能信息。映射 负责特定修饰事件的酶具有指导性，因为它将这些修饰放置在 指示生物功能的信号分子的背景。该中心的许多DBP将建立 将作为进一步研究的基础的临时技术措施的全面清单。为这些项目提供方向 研究，有针对性的，但可伸缩的，分析是必要的，以绘制调节修饰位点的酶 利息。

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.