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Carbocation Footprinting of Soluble and Transmembrane Proteins
Analytical Chemistry ( IF 7.4 ) Pub Date : 2021-09-24 , DOI: 10.1021/acs.analchem.1c03274
Jie Sun 1 , Shuang Li 2 , Weikai Li 2 , Michael L Gross 1
Affiliation  

Here, we introduce carbocations (R3C+) as laser-initiated footprinting reagents for proteins. We screened seven candidates and selected trifluomethoxy benzyl bromide (TFBB) as an effective precursor for the electrophilic trifluomethoxy benzyl carbocation (TFB+) under laser (248 nm) irradiation on the fast photochemical oxidation of proteins (FPOP) platform. Initial results demonstrate that this electrophilic cation reagent affords residue coverage of nucleophilic amino acids including H, W, M, and S. Further, the addition of TFB+ increases the hydrophobicity of the peptides so that separation of isomeric peptide products by reversed-phase LC is improved, suggesting opportunities for subresidue footprinting. Comparison of apo- and holo-myoglobin footprints shows that the TFB+ footprinting is sensitive to protein conformational change and solvent accessibility. Interestingly, because the TFB+ is amphiphilic, the reagent can potentially footprint membrane proteins as demonstrated for vitamin K epoxide reductase (VKOR) stabilized in a micelle. Not only does footprinting of the extra-membrane domain occur, but also some footprinting of the hydrophobic transmembrane domain is achieved owing to the interaction of TFB+ with the micelle. Carbocation precursors are stable and amenable for tailoring their properties and those of the incipient carbocation, enabling targeting their soluble or membrane-associated or embedded regions and distinguishing between the extra- and trans-membrane domains of membrane proteins.

中文翻译:

可溶性和跨膜蛋白的碳阳离子足迹

在这里,我们介绍了碳正离子 (R 3 C + ) 作为激光引发的蛋白质足迹试剂。我们筛选了七种候选物,并选择三氟甲氧基苄基溴 (TFBB) 作为蛋白质快速光化学氧化 (FPOP) 平台上激光 (248 nm) 照射下亲电三氟甲氧基苄基碳正离子 (TFB + )的有效前体。初步结果表明,这种亲电子阳离子试剂提供了对包括 H、W、M 和 S 在内的亲核氨基酸的残基覆盖。此外,添加 TFB +增加了肽的疏水性,从而改进了反相 LC 对异构肽产物的分离,这表明了亚残基足迹的机会。载脂蛋白和全息肌红蛋白足迹的比较表明,TFB +足迹对蛋白质构象变化和溶剂可及性敏感。有趣的是,由于 TFB +是两亲性的,因此该试剂可以潜在地覆盖膜蛋白,如稳定在胶束中的维生素 K 环氧化物还原酶 (VKOR) 所证明的那样。由于 TFB +的相互作用,不仅发生了膜外结构域的足迹,而且还实现了疏水性跨膜结构域的一些足迹。与胶束。碳正离子前体稳定且易于调整其性质和初始碳正离子的性质,从而能够靶向其可溶性或膜相关或嵌入区域并区分膜蛋白的膜外和跨膜结构域。
更新日期:2021-10-06
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