High-Resolution Mapping of Carbene-Based Protein Footprints
High-Resolution Mapping of Carbene-Based Protein Footprints
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DOI：
10.1021/ac300120z
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发表时间：
2012-05-15
影响因子：
7.4
通讯作者：
Schriemer, David C.
中科院分区：
文献类型：
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作者：
Jumper, Chanelle C.;Bomgarden, Ryan;Schriemer, David C.
卡宾化学最近已被用于结构质谱作为标记方法映射蛋白质表面。目前的研究提出了一种在氨基酸水平上定量标记分布的方法，并探讨了早期观察标记偏倚的性质和基础。通过使用基于液相色谱-串联质谱（LC-MS/MS）的方法应用于全钙调蛋白的消化物，我们开发了一种定量策略来绘制离子标记前体2-氨基-4，4-氮杂戊酸和4，4-氮杂戊酸光解产生的卡宾的位点特异性掺入。该方法提供了可靠的掺入数据的电子转移解离产生的片段，而高能量碰撞解离导致能量和序列依赖性的标签作为中性的损失。然而，这两种方法都可以产生适合于绘制全钙调蛋白与M13肽配体相互作用中残基的数据。位点特异性标记作为试剂，离子强度和温度的函数进行监测，表明在蛋白质表面的静电相互作用可以“引导”标签前体的分布到表面电荷的位点，并有利于标签插入到表面电荷附近的残基中。基于化学反应性，观察到插入羧酸酯的进一步偏好。我们建议，解耦表面分区从插入的化学提供了一个灵活的，可调的标记策略的结构质谱，可应用于广泛的蛋白质表面组成，并促进试剂的设计，以简化工作流程。
Carbene chemistry has been used recently in structural mass spectrometry as a labeling method for mapping protein surfaces. The current study presents a method for quantitating label distribution at the amino acid level and explores the nature and basis for an earlier observation of labeling bias. With the use of a method based on liquid chromatography-tandem mass spectrometry (LC-MS/MS) applied to digests of holo-calmodulin, we developed a quantitation strategy to map site-specific incorporation of carbene, generated from photolysis of ionic label precursors 2-amino-4,4-azipentanoic acid and 4,4-azipentanoic acid. The approach provides reliable incorporation data for fragments generated by electron-transfer dissociation, whereas high-energy collisional dissociation leads to energy and sequence-dependent loss of the label as a neutral. However, both can produce data suitable for mapping residues in the interaction of holo-calmodulin with M13 peptide ligand. Site-specific labeling was monitored as a function of reagent, ionic strength, and temperature, demonstrating that electrostatic interactions at the protein surface can "steer" the distribution of label precursors to sites of surface charge and favor label insertion into residues in the vicinity of the surface charge. A further preference for insertion into carboxylates was observed, based on chemical reactivity. We suggest that decoupling surface partitioning from the chemistry of insertion offers a flexible, tunable labeling strategy for structural mass spectrometry that can be applied to a broad range of protein surface compositions and promotes the design of reagents to simplify the workflow.