High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid,Journal of Proteome Research

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High-Performance Workflow for Identifying Site-Specific Crosslinks Originating from a Genetically Incorporated, Photoreactive Amino Acid
Journal of Proteome Research ( IF 3.8 ) Pub Date : 2024-07-05 , DOI: 10.1021/acs.jproteome.4c00194
Lindsey D Ulmer ₁ , Daniele Canzani ₁ , Christopher N Woods ₂ , Natalie L Stone ₂ , Maria K Janowska ₂ , Rachel E Klevit ₂ , Matthew F Bush ₁

Affiliation

In conventional crosslinking mass spectrometry, proteins are crosslinked using a highly selective, bifunctional chemical reagent, which limits crosslinks to residues that are accessible and reactive to the reagent. Genetically incorporating a photoreactive amino acid offers two key advantages: any site can be targeted, including those that are inaccessible to conventional crosslinking reagents, and photoreactive amino acids can potentially react with a broad range of interaction partners. However, broad reactivity imposes additional challenges for crosslink identification. In this study, we incorporate benzoylphenylalanine (BPA), a photoreactive amino acid, at selected sites in an intrinsically disordered region of the human protein HSPB5. We report and characterize a workflow for identifying and visualizing residue-level interactions originating from BPA. We routinely identify 30 to 300 crosslinked peptide spectral matches with this workflow, which is up to ten times more than existing tools for residue-level BPA crosslink identification. Most identified crosslinks are assigned to a precision of one or two residues, which is supported by a high degree of overlap between replicate analyses. Based on these results, we anticipate that this workflow will support the more general use of genetically incorporated, photoreactive amino acids for characterizing the structures of proteins that have resisted high-resolution characterization.

中文翻译：

用于识别源自基因整合光反应性氨基酸的位点特异性交联的高性能工作流程

在传统的交联质谱分析中，蛋白质使用高度选择性的双功能化学试剂进行交联，这将交联限制在可与试剂接触和反应的残基上。通过基因方式整合光反应性氨基酸具有两个关键优势：可以靶向任何位点，包括传统交联试剂无法到达的位点，并且光反应性氨基酸可以与广泛的相互作用伙伴发生反应。然而，广泛的反应性给交联识别带来了额外的挑战。在这项研究中，我们在人类蛋白质 HSPB5 本质上无序区域的选定位点掺入苯甲酰基苯丙氨酸 (BPA)（一种光反应性氨基酸）。我们报告并描述了用于识别和可视化源自 BPA 的残留水平相互作用的工作流程。我们通常使用此工作流程识别 30 至 300 个交联肽光谱匹配，这比现有的残留水平 BPA 交联识别工具多出十倍。大多数已识别的交联被指定为一或两个残基的精度，这是由重复分析之间的高度重叠所支持的。基于这些结果，我们预计该工作流程将支持更广泛地使用基因整合的光反应性氨基酸来表征难以进行高分辨率表征的蛋白质结构。

更新日期：2024-07-05

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