Progress toward Proteome-Wide Photo-Cross-Linking to Enable Residue-Level Visualization of Protein Structures and Networks In Vivo
Progress toward Proteome-Wide Photo-Cross-Linking to Enable Residue-Level Visualization of Protein Structures and Networks In Vivo
复制标题
DOI：
10.1021/acs.analchem.3c01369
复制
发表时间：
2023-06-21
影响因子：
7.4
通讯作者：
Fried，Stephen D. D.
中科院分区：
文献类型：
--
作者：
Faustino，Anneliese M. M.;Sharma，Piyoosh;Fried，Stephen D. D.
交联质谱 (XL-MS) 是结构生物学和细胞生物学十字路口的一种新兴方法，具有独特的能力，能够以残基水平分辨率和蛋白质组范围内识别蛋白质-蛋白质相互作用。随着交联剂的发展，交联剂可以在细胞内形成连接，并在质谱仪上的裂解过程中轻松裂解（MS 可裂解交联），识别复杂样品（包括活细胞或组织）中任意两种蛋白质之间的接触变得越来越容易。光交联剂具有高时间分辨率和高反应活性的优点，从而可以结合所有残基类型（而不仅仅是赖氨酸）；然而，光交联剂尚未得到广泛使用，并且尚未用于全蛋白质组研究，因为它们的产品难以识别。在这里，我们展示了两种异双功能光交联剂的合成和应用，其特征是二氮丙啶和N-羟基-琥珀酰亚胺基氨基甲酸酯基团，后者在酰基转移到蛋白质靶标时揭示了双裂变MS可裂解的连接。此外，这些交联剂表现出高水溶性和细胞渗透性。使用这些化合物，我们证明了纤维素中蛋白质组范围光交联的可行性。这些研究阐明了大肠杆菌相互作用网络的一小部分，尽管具有残留水平的分辨率。通过进一步优化，这些方法将能够在其天然环境中以残基水平分辨率检测蛋白质五元相互作用网络，我们预计它们将有助于探索细胞的分子社会学。
Cross-linking mass spectrometry (XL-MS) is emerging as a method at the crossroads of structural and cellular biology, uniquely capable of identifying protein–protein interactions with residue-level resolution and on the proteome-wide scale. With the development of cross-linkers that can form linkages inside cells and easily cleave during fragmentation on the mass spectrometer (MS-cleavable cross-links), it has become increasingly facile to identify contacts between any two proteins in complex samples, including in live cells or tissues. Photo-cross-linkers possess the advantages of high temporal resolution and high reactivity, thereby engaging all residue-types (rather than just lysine); nevertheless, photo-cross-linkers have not enjoyed widespread use and are yet to be employed for proteome-wide studies because their products are challenging to identify. Here, we demonstrate the synthesis and application of two heterobifunctional photo-cross-linkers that feature diazirines andN-hydroxy-succinimidyl carbamate groups, the latter of which unveil doubly fissile MS-cleavable linkages upon acyl transfer to protein targets. Moreover, these cross-linkers demonstrate high water-solubility and cell-permeability. Using these compounds, we demonstrate the feasibility of proteome-wide photo-cross-linkingin cellulo. These studies elucidate a small portion ofEscherichia coli’s interaction network, albeit with residue-level resolution. With further optimization, these methods will enable the detection of protein quinary interaction networks in their native environment at residue-level resolution, and we expect that they will prove useful toward the effort to explore the molecular sociology of the cell.