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首页> 外文会议>ASMS Conference on Mass Spectrometry and Allied Topics >Development of a Novel Multiplexed Quantitative Cross-linking Mass Spectrometry (QXL-MS) Strategy to Define the Structural Dynamics of Protein Complexes
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Development of a Novel Multiplexed Quantitative Cross-linking Mass Spectrometry (QXL-MS) Strategy to Define the Structural Dynamics of Protein Complexes

机译：开发一种新型多路复用定量​​交联质谱（QXL-MS）策略，以确定蛋白质复合物的结构动态

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Cross-linking mass spectrometry (XL-MS) represents a recently popularized hybrid strategy for probing subunit interactions within static protein assemblies and defining the interaction interfaces facilitating protein-protein interactions (PPIs). One of the major challenges in conventional XL-MS studies is the unambiguous identification of cross-linked peptides, due to difficulty in interpreting convoluted tandem mass spectra resulting from the fragmentation of covalently linked peptides. To this end, we have developed a new class of sulfoxide-containing MS-cleavable cross-linking reagents: i.e., disuccinimidyl sulfoxide (DSSO) {Kao, 2011; Kao, 2012}, dimethyl disuccinimidyl sulfoxide (DMDSSO) {Yu, 2014; Yu, 2015}, azide-tagged acid-cleavable disuccinimidyl bissulfoxide (Azide-A-DSBSO) {Kaake, 2014}. These MS-cleavable reagents enable simplified and unambiguous identification of cross-linked peptides by MS~n analysis and conventional database searching tools. To determine protein structural dynamics, we developed the d_0/d_(10)-DMDSSO based quantitative XL-MS (QXL-MS) strategy to discern the conformational changes of subunits within multi-protein complexes in response to various forms of stimuli {Yu, 2014; Yu, 2015}. However, these approaches are limited by chemical derivatization of cross-linking reagents, restricting their application to pair-wise (side-by-side) comparisons of structural and topological changes. In order to allow a multiplex-able comparison of protein structures under different conditions simultaneously, we have developed a novel QXL-MS strategy by integrating TMT reagents and sulfoxide-containing MS-cleavable reagents. This strategy represents the first application of TMT reagents for multiplexing quantitation of cross-linked peptides and will significantly facilitate multiplexed quantitative analysis of the conformational dynamics of protein complexes using cross-linking mass spectrometry.
机译：交联质谱（XL-MS）表示用于探测静态蛋白组件内亚基相互作用并限定的交互接口促进蛋白质 - 蛋白质相互作用（质子泵抑制剂）最近普及混合策略。之一在常规XL-MS研究的主要挑战是交联的肽的明确识别，由于在解释从共价连接的肽的断裂产生卷积串联质谱困难。为此，我们已经开发了一类新的含亚砜-MS-裂解的交联试剂的：即，二琥珀酰亚胺砜（DSSO）{花王，2011;花王，2012}，二甲基琥珀酰亚胺基亚砜（DMDSSO）{于，2014;于，2015}，叠氮化物标记的酸可裂解的二琥珀酰亚胺bissulfoxide（叠氮基的A-DSBSO）{Kaake，2014}。这些MS裂解试剂使能简化，并通过MS〜n的分析和常规数据库的检索工具的交联的肽的明确识别。为了测定蛋白结构动力学，我们开发了D_0 / D_（10）基于-DMDSSO定量XL-MS（QXL-MS）策略来辨别响应于各种形式的刺激{于多蛋白复合物之内亚基的构象变化， 2014;玉，2015年}。然而，这些方法是通过交联试剂的化学衍生化的限制，限制了它们的应用程序的结构和拓扑结构变化成对（并排侧）比较。为了同时允许蛋白质结构的不同条件下的多重比较能，我们通过整合TMT试剂和含有亚砜-MS裂解试剂开发了一种新颖QXL-MS策略。该策略代表TMT试剂的用于复用的交联的肽的定量所述第一应用和将显著有助于使用交联质谱蛋白质复合物的构象动力学的复用定量分析。

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