Department of Chemistry and Biochemistry, and Department of Biological Chemistry, University of California-Los Angeles , Los Angeles, California 90095, United States.
Discovery Analytical Sciences, Amgen , South San Francisco, California 94080, United States.
Anal Chem. 2016 Dec 20;88(24):12427-12436. doi: 10.1021/acs.analchem.6b03762. Epub 2016 Dec 1.
Over the past two decades, orthogonal acceleration time-of-flight has been the de facto analyzer for solution and membrane-soluble protein native mass spectrometry (MS) studies; this however is gradually changing. Three MS instruments are compared, the Q-ToF, Orbitrap, and the FT-ICR, to analyze, under native instrument and buffer conditions, the seven-transmembrane helical protein bacteriorhodopsin-octylglucoside micelle and the empty nanodisc (MSP1D1-Nd) using both MS and tandem-MS modes of operation. Bacteriorhodopsin can be released from the octylglucoside-micelle efficiently on all three instruments (MS-mode), producing a narrow charge state distribution (z = 8+ to 10+) by either increasing the source lens or collision cell (or HCD) voltages. A lower center-of-mass collision energy (0.20-0.41 eV) is required for optimal bacteriorhodopsin liberation on the FT-ICR, in comparison to the Q-ToF and Orbitrap instruments (0.29-2.47 eV). The empty MSP1D1-Nd can be measured with relative ease on all three instruments, resulting in a highly complex spectrum of overlapping, polydisperse charge states. There is a measurable difference in MSP1D1-Nd charge state distribution (z = 15+ to 26+), average molecular weight (141.7 to 169.6 kDa), and phospholipid incorporation number (143 to 184) under low activation conditions. Utilizing tandem-MS, bacteriorhodopsin can be effectively liberated from the octylglucoside-micelle by collisional (Q-ToF and FT-ICR) or continuous IRMPD activation (FT-ICR). MSP1D1-Nd spectral complexity can also be significantly reduced by tandem-MS (Q-ToF and FT-ICR) followed by mild collisional or continuous IRMPD activation, resulting in a spectrum in which the charge state and phospholipid incorporation levels can easily be determined.
在过去的二十年中，正交加速飞行时间已成为溶液和膜溶性蛋白质天然质谱（MS）研究的实际分析器；但这种情况正在逐渐改变。本文比较了 Q-ToF、Orbitrap 和 FT-ICR 三种 MS 仪器，在天然仪器和缓冲条件下，使用 MS 和串联-MS 操作模式，分析七跨膜螺旋蛋白菌紫质-辛基葡糖苷胶束和空纳米盘（MSP1D1-Nd）。在所有三种仪器上（MS 模式），都可以有效地从辛基葡糖苷胶束中释放菌紫质，通过增加源透镜或碰撞池（或 HCD）电压，可以产生窄的电荷状态分布（z = 8+ 到 10+）。与 Q-ToF 和 Orbitrap 仪器相比（0.29-2.47 eV），FT-ICR 仪器上菌紫质的最佳释放需要较低的质心碰撞能量（0.20-0.41 eV）。在所有三种仪器上，都可以相对容易地测量空 MSP1D1-Nd，产生高度复杂的重叠多分散电荷状态谱。在低激活条件下，MSP1D1-Nd 的电荷状态分布（z = 15+ 到 26+）、平均分子量（141.7 到 169.6 kDa）和磷脂掺入数（143 到 184）有可测量的差异。利用串联-MS，菌紫质可以通过碰撞（Q-ToF 和 FT-ICR）或连续 IRMPD 激活（FT-ICR）有效地从辛基葡糖苷胶束中释放出来。串联-MS（Q-ToF 和 FT-ICR）后再进行温和的碰撞或连续的 IRMPD 激活，也可以显著降低 MSP1D1-Nd 光谱的复杂性，得到一个易于确定电荷状态和磷脂掺入水平的光谱。
