Chemical cross-linking is a powerful strategy for elucidating the structures of protein or protein complexes. The distance constraints obtained from cross-linked peptides represent the three-dimensional structures of the protein complexes. Unfortunately, structural analysis using cross-linking approach demands a significant amount of data to elucidate protein structures. This requires the development of several cleavable cross-linkers with different range of spacer chains. An Electron Transfer Dissociation (ETD) tandem mass spectrometry cleavable bond hydrazone was reported. Its fragmentation with conjugated peptides showed promise for the development of a new ETD cleavable cross-linker. However, no cross-linker was developed utilizing this ETD cleavable bond. For the first time, we attempted to develop an ETD cleavable cross-linker utilizing a hydrazone bond. We overcome the pitfall for the synthesis of this cross-linker and an easy synthesis scheme is reported. In this report, we evaluated the performance of this cross-linker called Hydrazone Incorporated ETD cleavable cross-linker (HI-ETD-XL) in model peptides and proteins. The characteristic fragmentation behavior of HI-ETD-XL during electron transfer dissociation and subsequent sequence identification of the peptide fragment ions by tandem mass spectrometry allowed the identification of cross-linked peptides unambiguously. We believe the availability of this ETD cleavable cross-linker will advance structural proteomics research significantly.

Significance

Many cellular processes rely on the structural dynamics of protein complexes. The detailed knowledge of the structure and dynamics of protein complexes is crucial for understanding their biological functions and regulations. However, most of the structure of these multiprotein entities remain uncharacterized and sometimes is very challenging to reveal with biophysical techniques alone. Chemical cross-linking combined with mass spectrometry (MS) has proven to be a dependable strategy in structural proteomics field. However, data complexity and false identifications are significant hindrances for unambiguous identification of cross-linked peptides. Confident identifications demand structural studies with cross-linkers with different properties and variable spacer chain lengths. This new ETD cleavable cross-linking workflow will provide additional confidence to overcome these drawbacks and allow us to pinpoint cross-linked peptides confidently.

中文翻译：

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评估用于阐明蛋白质结构的 ETD 可裂解交联策略的性能。

化学交联是阐明蛋白质或蛋白质复合物结构的有力策略。从交联肽获得的距离约束代表了蛋白质复合物的三维结构。不幸的是，使用交联方法进行结构分析需要大量数据来阐明蛋白质结构。这需要开发具有不同间隔链范围的几种可裂解交联剂。报道了电子转移解离 (ETD) 串联质谱可裂解键腙。其与偶联肽的片段化显示出开发新的 ETD 可切割交联剂的前景。然而，没有使用这种 ETD 可裂解键开发交联剂。首次，我们试图开发一种利用腙键的 ETD 可裂解交联剂。我们克服了合成这种交联剂的陷阱，并报告了一个简单的合成方案。在本报告中，我们评估了这种称为 Hydrazone Incorporated ETD 可裂解交联剂 (HI-ETD-XL) 的交联剂在模型肽和蛋白质中的性能。HI-ETD-XL 在电子转移解离过程中的特征性碎裂行为和随后通过串联质谱法对肽碎片离子的序列鉴定允许明确地鉴定交联肽。我们相信这种 ETD 可裂解交联剂的可用性将显着推进结构蛋白质组学研究。在本报告中，我们评估了这种称为 Hydrazone Incorporated ETD 可裂解交联剂 (HI-ETD-XL) 在模型肽和蛋白质中的交联剂的性能。HI-ETD-XL 在电子转移解离过程中的特征性碎裂行为和随后通过串联质谱法对肽碎片离子的序列鉴定允许明确鉴定交联肽。我们相信这种 ETD 可裂解交联剂的可用性将显着推进结构蛋白质组学研究。在本报告中，我们评估了这种称为 Hydrazone Incorporated ETD 可裂解交联剂 (HI-ETD-XL) 在模型肽和蛋白质中的交联剂的性能。HI-ETD-XL 在电子转移解离过程中的特征性碎裂行为和随后通过串联质谱法对肽碎片离子的序列鉴定允许明确地鉴定交联肽。我们相信这种 ETD 可裂解交联剂的可用性将显着推进结构蛋白质组学研究。HI-ETD-XL 在电子转移解离过程中的特征性碎裂行为和随后通过串联质谱法对肽碎片离子的序列鉴定允许明确地鉴定交联肽。我们相信这种 ETD 可裂解交联剂的可用性将显着推进结构蛋白质组学研究。HI-ETD-XL 在电子转移解离过程中的特征性碎裂行为和随后通过串联质谱法对肽碎片离子的序列鉴定允许明确地鉴定交联肽。我们相信这种 ETD 可裂解交联剂的可用性将显着推进结构蛋白质组学研究。

意义

许多细胞过程依赖于蛋白质复合物的结构动力学。蛋白质复合物的结构和动力学的详细知识对于理解它们的生物学功能和调节至关重要。然而，这些多蛋白实体的大部分结构仍未表征，有时仅用生物物理技术来揭示是非常具有挑战性的。化学交联结合质谱 (MS) 已被证明是结构蛋白质组学领域的可靠策略。然而，数据复杂性和错误识别是明确识别交联肽的重大障碍。可靠的鉴定需要使用具有不同特性和可变间隔链长度的交联剂进行结构研究。