Department of Chemistry, University of Washington, P.O. Box 351700, Seattle, Washington 98195-1700, United States.
Department of Biochemistry, University of Washington, P.O. Box 357350, Seattle, Washington 98195-7350, United States.
J Proteome Res. 2024 Aug 2;23(8):3560-3570. doi: 10.1021/acs.jproteome.4c00194. Epub 2024 Jul 5.
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.
在传统的交联质谱中，蛋白质是使用高度选择性的双功能化学试剂交联的，这将交联限制在对试剂可及和反应的残基上。通过遗传方式引入光反应性氨基酸具有两个关键优势：任何部位都可以成为目标，包括那些传统交联试剂不可及的部位，并且光反应性氨基酸可以潜在地与广泛的相互作用伙伴发生反应。然而，广泛的反应性为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 HSPB5 的一个固有无序区域的选定部位引入了苯甲酰苯丙氨酸（BPA），一种光反应性氨基酸。我们报告并描述了一种用于识别和可视化源自 BPA 的残基水平相互作用的工作流程。我们通常使用这种工作流程可以识别 30 到 300 个交联肽谱匹配，这比现有的用于残基水平 BPA 交联识别的工具多十倍。大多数鉴定的交联都被分配到一个或两个残基的精度，这得到了重复分析之间高度重叠的支持。基于这些结果，我们预计这种工作流程将支持更广泛地使用遗传引入的光反应性氨基酸来表征那些难以进行高分辨率表征的蛋白质的结构。
