Increased Coverage in the Transmembrane Domain with Activated-Ion Electron Capture Dissociation for Top-Down Fourier-Transform Mass Spectrometry of Integral Membrane Proteins

The c-subunit of ATP synthase (AtpH) is an 8 kD integral membrane protein with two transmembrane domains; we set out to demonstrate it amenable to top-down electrospray-ionization Fourier-transform mass spectrometry (FT−MS) using both collision activated and electron capture dissociation (CAD/ECD). Thermal activation concomitant with electron delivery was necessary for efficient ECD (activated-ion ECD; aiECD), yielding complementary information and greater sequence coverage in the transmembrane domains in comparison with CAD. Keywords: electrospray-ionization mass spectrometry (ESI-MS) • Fourier-transform mass spectrometry (FT−MS) • Fourier-transform ion cyclotron resonance (FTICR) • collision activated dissociation (CAD) • electron capture dissociation (ECD) • activated-ion ECD (aiECD) • infrared multiphoton dissociation (IRMPD) • thylakoid membrane • membrane protein • ATP synthase

ATP合酶的c亚基（AtpH）是一种分子量为8千道尔顿的整合膜蛋白（integral membrane protein），包含两个跨膜结构域（transmembrane domains）。本研究旨在验证其可适用于采用碰撞活化解离（collision activated dissociation, CAD）与电子捕获解离（electron capture dissociation, ECD）两种解离模式的自上而下电喷雾电离傅里叶变换质谱（Fourier-transform mass spectrometry, FT−MS）分析。相较于碰撞活化解离，热活化与电子递送协同作用对于实现高效电子捕获解离（活化离子电子捕获解离，activated-ion ECD, aiECD）而言是必要的，该方法可获得互补的质谱信息，并在跨膜结构域中实现更高的序列覆盖度。关键词：电喷雾电离质谱（electrospray-ionization mass spectrometry, ESI-MS） • 傅里叶变换质谱（Fourier-transform mass spectrometry, FT−MS） • 傅里叶变换离子回旋共振质谱（Fourier-transform ion cyclotron resonance, FTICR） • 碰撞活化解离（collision activated dissociation, CAD） • 电子捕获解离（electron capture dissociation, ECD） • 活化离子电子捕获解离（activated-ion ECD, aiECD） • 红外多光子解离（infrared multiphoton dissociation, IRMPD） • 类囊体膜（thylakoid membrane） • 膜蛋白 • ATP合酶