Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.
Infrared Multiphoton Dissociation Enables Top-Down Characterization of Membrane Protein Complexes and G Protein-Coupled Receptors.
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DOI：
10.1002/anie.202305694
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发表时间：
2023-09-04
期刊：
影响因子：
--
通讯作者：
Robinson CV
中科院分区：
文献类型：
--
作者：
Lutomski CA;El-Baba TJ;Hinkle JD;Liko I;Bennett JL;Kalmankar NV;Dolan A;Kirschbaum C;Greis K;Urner LH;Kapoor P;Yen HY;Pagel K;Mullen C;Syka JEP;Robinson CV
膜蛋白具有天然质谱分析的挑战性，因为它们的疏水性通常需要在分析之前通过碰撞激活去除洗涤剂胶束。然而，可以应用的能量量存在实际限制，这通常妨碍了自上而下的质谱分析的后续表征。为了克服这一障碍，我们在高压线性离子阱中应用了一种改进的Orbitrap Eclipse Tribrid质谱仪，该质谱仪与红外激光器耦合。我们展示了如何调整入射光子的强度和时间，使膜蛋白从洗涤剂胶束中解放出来。具体地说，我们将胶束去除的难易性与洗涤剂在凝聚相和气相中的红外吸收联系起来。通过红外多光子解离（IRMPD）进行自上而下的质谱分析，可以获得良好的序列覆盖率，从而明确识别膜蛋白及其复合物。通过对比和比较氨通道与两种A类gpcr的断裂模式，我们确定了跨膜结构域内相邻氨基酸的连续切割。通过气相分子动力学模拟，我们发现在温度升高的情况下，容易破碎的区域保持了蛋白质结构的各个方面。总之，我们提出了一个基本原理来解释为什么和在哪里蛋白质片段离子产生。
Membrane proteins are challenging to analyze by native mass spectrometry (MS) as their hydrophobic nature typically requires detergent micelles that are removed prior to analysis via collisional activation. There is however a practical limit to the amount of energy which can be applied, which often precludes subsequent characterization by top-down MS. To overcome this barrier, we have applied a modified Orbitrap Eclipse Tribrid mass spectrometer coupled to an infrared laser within a high-pressure linear ion trap. We show how tuning the intensity and time of incident photons enables liberation of membrane proteins from detergent micelles. Specifically, we relate the ease of micelle removal to the infrared absorption of detergents in both condensed and gas phases. Top-down MS via infrared multiphoton dissociation (IRMPD), results in good sequence coverage enabling unambiguous identification of membrane proteins and their complexes. By contrasting and comparing the fragmentation patterns of the ammonia channel with two class A GPCRs, we identify successive cleavage of adjacent amino acids within transmembrane domains. Using gas-phase molecular dynamics simulations, we show that areas prone to fragmentation maintain aspects of protein structure at increasing temperatures. Altogether, we propose a rationale to explain why and where in the protein fragment ions are generated.
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完整膜蛋白复合物的质谱法。
影响因子：
14.8
作者：
通讯作者：
--
DOI：
10.1007/978-1-0716-1585-0_3
发表时间：
2022-01-01
期刊：
Methods in molecular biology (Clifton, N.J.)
影响因子：
--
作者：
Cong, Xiao;Patrick, John W;Laganowsky, Arthur
通讯作者：
Laganowsky, Arthur
影响因子：
7.4
作者：
Keener JE;Zhang G;Marty MT
通讯作者：
Marty MT
影响因子：
7.4
作者：
Kumar, Smriti;Zhu, Yun;Stover, Lauren;Laganowsky, Arthur
通讯作者：
Laganowsky, Arthur
影响因子：
3.4
作者：
Gimpelev, M;Forrest, LR;Honig, B
通讯作者：
Honig, B