Department of Chemistry, Texas A&M University, College Station, Texas 77843, United States.
J Am Soc Mass Spectrom. 2023 May 3;34(5):977-980. doi: 10.1021/jasms.3c00071. Epub 2023 Mar 31.
The analysis of large molecules is challenging, as they often have salts and adducts retained through the electrospray process, which increase the observed mass and compromise the achievable mass resolution. Mild collisional activation has been shown to be very effective for the removal of adducts and increases both measurement accuracy and mass resolution of large (>100 kDa) protein complexes. Collisionally activated protein ions are more completely desolvated due to the increased number of collisions when trapped following activation. A short square quadrupole maintained at 300 mTorr by a mechanical pump was added between the ion funnel and transmission quadrupole. This configuration and operation effectively removed adducts from the 800 kDa tetradecamer GroEL as well as fragmented smaller protein complexes like C-reactive protein. Due to the gas high pressure, ions of low size-to-charge ratio, such as those in charge reducing buffers, had low ejection efficiency. We show that segmenting the quadrupole rods greatly improves signal intensity for charge reduced GroEL D398A mutant compared to nonsegmented rods when operating at high pressure.
分析大分子具有挑战性，因为它们通常通过电喷雾过程保留盐和加合物，这会增加观察到的质量并影响可实现的质量分辨率。温和的碰撞激活已被证明对于去除加合物非常有效，并且可以提高大（> 100 kDa）蛋白质复合物的测量精度和质量分辨率。由于在激活后被捕获时增加了碰撞次数，因此碰撞激活的蛋白质离子更完全地去溶剂化。通过机械泵将一个短的方形四极杆保持在 300 mTorr 的压力下，添加在离子阱和传输四极杆之间。这种配置和操作有效地从 800 kDa 十四聚体 GroEL 中去除了加合物，并且还使较小的蛋白质复合物（如 C 反应蛋白）发生了碎片化。由于气体压力高，电荷减小缓冲液等低电荷质量比的离子的引出效率较低。我们表明，与非分段杆相比，当在高压下操作时，分段四极杆棒可大大提高电荷还原型 GroEL D398A 突变体的信号强度。
