Administrative Supplement to Characterizing proteasome-substrate interactions by mass spectrometry proteomics

项目总结 我们建议购买一台Thermo FAIMS Pro Orbitrap离子源，它将连接到前面 并用于我们的泛素-蛋白酶体交联实验。我们的研究 该项目涉及探索多泛素链、蛋白酶体模型底物和 酵母菌蛋白酶体。交联质谱学正被应用于发现新的泛素 为了更好地了解标记底物如何影响蛋白酶体功能。这是至关重要的 由于生化分析的复杂性，这种交联链的鉴定具有高度的可信度 建立在这些结果的基础上。由于这一领域对于什么构成 明确的交叉链接识别，我们正在做出相当大的努力来改进交叉链接 观察和解释方法论。通过结合电子转移解离、碰撞 前驱离子的诱导解离和高能碰撞诱导解离 报告了如何提高交叉链接标识的可信度。对这一项目的成功至关重要 方法是有强烈的前体离子信号。Thermo FAIMS Pro轨道俘获离子源是 旨在增加高电荷态离子的传输，如那些与交叉离子相关的离子 连接的多肽，并区分背景物种，如个别多肽。事实上，它 已经在交联质谱学实验中证明了成功。我们将使用这个 发现和确认涉及多聚泛素和酵母蛋白酶体的交联链的装置 这样做将实现分析和生物学方面的进步。我们识别和识别的能力取得了进展 最终确定交联肽对于阐明一些最重要的 蛋白质科学中的重要和具有挑战性的问题。具体地说，我们的交叉连接蛋白质组学 实验将为蛋白酶体上的新底物受体提供候选，并将识别 多聚泛素中对蛋白酶体识别最有效的大小和连接模式。 总而言之，这个项目应该会在交叉连接方法和我们的 了解蛋白酶体如何识别和消化底物。

PROJECT SUMMARY We are proposing to purchase a Thermo FAIMS Pro Orbitrap Ion Source that will attach to the front of a Lumos Orbitrap and be used in our ubiquitin-proteasome cross-linking experiments. Our research project involves probing interactions between polyubiquitin chains, model proteasome substrates and the yeast proteasome. Cross-linking mass spectrometry is being applied to discover novel ubiquitin receptors and to better understand how tagged substrates can impact proteasome function. It is crucial that cross-links are identified with high confidence because of the complexity of the biochemical assays that build on these results. Since there is limited consensus in this field as to what constitutes an unambiguous cross-link identification, we are exerting considerable effort to improve cross-linking observation and interpretation methodology. By combining electron transfer dissociation, collision induced dissociation, and high energy collision induced dissociation of precursor ions we have recently reported how the credibility of cross-link identifications can be enhanced. Crucial to the success of this approach is to have intense precursor ion signals. The Thermo FAIMS Pro Orbitrap Ion Source is designed to increase the transmission of highly-charged ions such as those associated with cross- linked peptides and to discriminate against background species such as individual peptides. In fact, it has already demonstrated success in cross-linking mass spectrometry experiments. We will use this device to discover and confirm cross-links involving polyubiquitin and the yeast proteasome and by doing so will achieve both analytical and biological advances. Progress in our ability to recognize and conclusively identify cross-linked peptides has transformative potential for elucidating some of the most important and challenging issues in protein science. Specifically, our cross-linking proteomics experiments will provide candidates for novel substrate receptors on the proteasome and will identify the sizes and linkage patterns in polyubiquitin that are most effective for recognition by the proteasome. In summary, this project should result in significant advances in cross-linking methodology and in our understanding of how the proteasome recognizes and digests substrates.