Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.
Serum Albumin Domain Structures in Human Blood Serum by Mass Spectrometry and Computational Biology.
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DOI：
10.1074/mcp.m115.048504
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发表时间：
2016-03
期刊：
影响因子：
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通讯作者：
Rappsilber J
中科院分区：
文献类型：
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作者：
Belsom A;Schneider M;Fischer L;Brock O;Rappsilber J
化学交联与质谱相结合已被证明可用于研究蛋白质-蛋白质相互作用和蛋白质结构，但交联数据的低密度迄今为止阻碍了其在从头确定结构中的应用。交联密度通常受到常用于蛋白质交联的标准交联试剂的化学选择性的限制。我们使用了一种异双功能交联剂，磺基琥珀酰亚胺基 4,4'-azipentanoate (sulfo-SDA)，结合了传统的磺基-N-羟基琥珀酰亚胺 (sulfo-NHS) 酯和 UV 光活化二氮丙啶基团。这种二氮丙啶产生高反应性和混杂的卡宾物种，与同型双功能、基于 NHS 的交联剂相比，最终结果是交联数量大大增加。我们提出了一种新颖的方法，将这种高密度光交联数据与构象空间搜索相结合，以研究来自纯化样品及其天然环境中人血清的人血清白蛋白结构域的结构。我们的方法能够以良好的精度确定人血清白蛋白结构域结构：对于第一个选定的结构，晶体结构的均方根偏差为 2.8/5.6/2.9 Å（纯化样品）和 4.5/5.9/4.8Å（血清样品）； 2.5/4.9/2.9 Å（纯化样品）和 3.5/5.2/3.8 Å（血清样品）是前五个选定结构中最好的。我们对人血清白蛋白的概念验证研究表明，我们的方法在确定更多蛋白质在复杂的生物环境中的结构方面具有初步潜力，这些蛋白质在这些环境中发挥作用，并且它们可能需要正确折叠。数据可通过 ProteomeXchange 获得，标识符为 PXD001692。
Chemical cross-linking combined with mass spectrometry has proven useful for studying protein-protein interactions and protein structure, however the low density of cross-link data has so far precluded its use in determining structures de novo. Cross-linking density has been typically limited by the chemical selectivity of the standard cross-linking reagents that are commonly used for protein cross-linking. We have implemented the use of a heterobifunctional cross-linking reagent, sulfosuccinimidyl 4,4′-azipentanoate (sulfo-SDA), combining a traditional sulfo-N-hydroxysuccinimide (sulfo-NHS) ester and a UV photoactivatable diazirine group. This diazirine yields a highly reactive and promiscuous carbene species, the net result being a greatly increased number of cross-links compared with homobifunctional, NHS-based cross-linkers. We present a novel methodology that combines the use of this high density photo-cross-linking data with conformational space search to investigate the structure of human serum albumin domains, from purified samples, and in its native environment, human blood serum. Our approach is able to determine human serum albumin domain structures with good accuracy: root-mean-square deviation to crystal structure are 2.8/5.6/2.9 Å (purified samples) and 4.5/5.9/4.8Å (serum samples) for domains A/B/C for the first selected structure; 2.5/4.9/2.9 Å (purified samples) and 3.5/5.2/3.8 Å (serum samples) for the best out of top five selected structures. Our proof-of-concept study on human serum albumin demonstrates initial potential of our approach for determining the structures of more proteins in the complex biological contexts in which they function and which they may require for correct folding. Data are available via ProteomeXchange with identifier PXD001692.