Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.
Characterization of protein cross-links via mass spectrometry and an open-modification search strategy.
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DOI：
10.1021/ac801646f
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发表时间：
2008-11-15
影响因子：
7.4
通讯作者：
Goodlett, David R.
中科院分区：
文献类型：
--
作者：
Singh, Pragya;Shaffer, Scott A.;Scherl, Alexander;Holman, Carol;Pfuetzner, Richard A.;Freeman, Theodore J. Larson;Miller, Samuel I.;Hernandez, Patricia;Appel, Ron D.;Goodlett, David R.
蛋白质-蛋白质相互作用是许多生物途径的功能和调节的关键。为了促进使用质谱法表征蛋白质-蛋白质相互作用，设计了一种新的数据采集/分析管道。该管道的目标是提供一种通用策略，用于从单个 LC/MS/MS 数据集中识别交联肽，而无需使用专门的交联剂或定制编写的软件。为了实现这一目标，交联肽对中的每个肽被认为是在未知氨基酸处用未知质量进行“翻译后”修饰。这允许使用开放修饰搜索引擎 Popitam 来解释交联肽的串联质谱。通过以高质量准确度获取前体和碎片，可以减少误报并提高数据库选择性。此外，还利用高电荷态驱动的数据采集方案来丰富交联肽的数据集。这种基于开放修饰搜索的管道被证明对于表征蛋白质中的化学交联和天然交联非常有用。通过表征化学交联 CYP2E1-b5 复合物中已知的相互作用来验证该管道。通过绘制 RcsF（一种参与 Rcs 磷酸传递的外膜脂蛋白）中的二硫桥，证明了该方法在识别天然交联方面的实用性。
Protein-protein interactions are key to function and regulation of many biological pathways. To facilitate characterization of protein-protein interactions using mass spectrometry, a new data acquisition/analysis pipeline was designed. The goal for this pipeline was to provide a generic strategy for identifying crosslinked peptides from single LC/MS/MS datasets, without using specialized crosslinkers or custom-written software. To achieve this, each peptide in the pair of crosslinked peptides was considered to be “post-translationally” modified with an unknown mass at an unknown amino acid. This allowed use of an open-modification search engine, Popitam, to interpret the tandem mass spectra of crosslinked peptides. False positives were reduced and database selectivity increased by acquiring precursors and fragments at high mass accuracy. Additionally, a high-charge-state-driven data acquisition scheme was utilized to enrich datasets for crosslinked peptides. This open-modification search based pipeline was shown to be useful for characterizing both chemical as well as native crosslinks in proteins. The pipeline was validated by characterizing the known interactions in chemically crosslinked CYP2E1-b5 complex. Utility of this method in identifying native crosslinks was demonstrated by mapping disulfide bridges in RcsF, an outer membrane lipoprotein involved in Rcs phosphorelay.
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