Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment.
Revealing ligand binding sites and quantifying subunit variants of noncovalent protein complexes in a single native top-down FTICR MS experiment.
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DOI：
10.1007/s13361-014-0928-6
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发表时间：
2014-12
影响因子：
3.2
通讯作者：
Loo, Joseph A.
中科院分区：
文献类型：
--
作者：
Li, Huilin;Wongkongkathep, Piriya;Van Orden, Steve L.;Loo, Rachel R. Ogorzalek;Loo, Joseph A.
关键词：
“原生”质谱（MS）已被证明在大分子组装体的结构生物学研究中越来越有用。以马肝酒精脱氢酶（hADH）和酵母酒精脱氢酶（yADH）为例，证明了傅里叶变换离子回旋质谱（FTICR MS）在一次天然自上而下质谱实验中可以获得丰富的信息。除了测量蛋白质复合物的分子量外，酵母ADH四聚体（147 kDa）在吸收模式下的平均分辨率为412,700 (m/z 5466)，质量反映了每个亚基结合两个锌原子。通过自上而下的电子捕获解离（ECD）实验对其n端89个氨基酸残基进行了测序，同时鉴定了Cys46上的锌结合位点和一个点突变（V58T）。结合多种激活/解离技术，包括ECD、源内解离（ISD）、碰撞激活解离（CAD）和红外多光子解离（IRMPD）， 40%的yADH序列直接来自天然四聚体配合物。对于hADH，原生自顶向下ECD-MS显示，在hADH样本中同时存在E和S亚基，其相对比例为4:1。天然自顶向下ISD MS hADH二聚体表明，每个亚基（E链和S链）不仅与两个锌原子结合，而且与NAD+/NADH配体结合，相对于E链，S链具有更高的NAD+/NADH结合偏好。E链和S链的序列覆盖率均达到32%。
“Native” mass spectrometry (MS) has been proven increasingly useful for structural biology studies of macromolecular assemblies. Using horse liver alcohol dehydrogenase (hADH) and yeast alcohol dehydrogenase (yADH) as examples, we demonstrate that rich information can be obtained in a single native top-down MS experiment using Fourier transform ion cyclotron mass spectrometry (FTICR MS). Beyond measuring the molecular weights of the protein complexes, isotopic mass resolution was achieved for yeast ADH tetramer (147 kDa) with an average resolving power of 412,700 at m/z 5466 in absorption mode and the mass reflects that each subunit binds to two zinc atoms. The N-terminal 89 amino acid residues were sequenced in a top-down electron capture dissociation (ECD) experiment, along with the identifications of the zinc binding site at Cys46 and a point mutation (V58T). With the combination of various activation/dissociation techniques, including ECD, in-source dissociation (ISD), collisionally activated dissociation (CAD), and infrared multiphoton dissociation (IRMPD), 40% of the yADH sequence was derived directly from the native tetramer complex. For hADH, native top-down ECD-MS shows that both E and S subunits are present in the hADH sample, with a relative ratio of 4:1. Native top-down ISD MS hADH dimer shows that each subunit (E and S chain) binds not only to two zinc atoms, but also the NAD+/NADH ligand, with a higher NAD+/NADH binding preference for the S chain relative to the E chain. In total, 32% sequence coverage was achieved for both E and S chains.
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DOI：
10.1039/c1an15286f
发表时间：
2011-10-07
期刊：
The Analyst
影响因子：
--
作者：
Cui W;Rohrs HW;Gross ML
通讯作者：
Gross ML
影响因子：
7.4
作者：
Benesch, JLP;Sobott, F;Robinson, CV
通讯作者：
Robinson, CV
影响因子：
16.6
作者：
Breuker, Kathrin;Brueschweiler, Sven;Tollinger, Martin
通讯作者：
Tollinger, Martin
影响因子：
48
作者：
Heck, Albert J. R.
通讯作者：
Heck, Albert J. R.
影响因子：
7.4
作者：
Li, Huilin;Lin, Tzu-Yung;Van Orden, Steve L.;Zhao, Yao;Barrow, Mark P.;Pizarro, Ana M.;Qi, Yulin;Sadler, Peter J.;O'Connor, Peter B.
通讯作者：
O'Connor, Peter B.