Department of Chemistry and Biochemistry, The University of Texas at Arlington, Arlington, TX, 76019, USA.
Shimadzu Scientific Instruments, Inc., Columbia, MD, USA.
J Am Soc Mass Spectrom. 2017 Sep;28(9):1977-1986. doi: 10.1007/s13361-017-1696-x. Epub 2017 May 30.
Recently, direct intact protein quantitation using triple quadrupole mass spectrometry (QqQ-MS) and multiple reaction monitoring (MRM) was demonstrated (J. Am. Soc. Mass Spectrom. 27, 886-896 (2016)). Even though QqQ-MS is known to provide extraordinary detection sensitivity for quantitative analysis, we found that intact proteins exhibited a less than 5% ion transmission from the first quadrupole to the third quadrupole mass analyzer in the presence of zero collision energy (ZCE). With the goal to enhance intact protein quantitation sensitivity, ion scattering effects, proton transfer effects, and mass filter resolution widths were examined for their contributions to the lost signal. Protein standards myoglobin and ubiquitin along with small molecules reserpine and vancomycin were analyzed together with various collision induced dissociation (CID) gases (N, He, and Ar) at different gas pressures. Mass resolution settings played a significant role in reducing ion transmission signal. By narrowing the mass resolution window by 0.35 m/z on each side, roughly 75%-90% of the ion signal was lost. The multiply charged proteins experienced additional proton transfer effects, corresponding to 10-fold signal reduction. A study of increased sensitivity of the method was also conducted with various MRM summation techniques. Although the degree of enhancement was analyte-dependent, an up to 17-fold increase in sensitivity was observed for ubiquitin using a summation of multiple MRM transitions. Biological matrix, human urine, and equine plasma were spiked with proteins to demonstrate the specificity of the method. This study provides additional insight into optimizing the use and sensitivity of QqQ-MS for intact protein quantification. Graphical Abstract ᅟ.
最近，使用三重四极杆质谱（QqQ-MS）和多重反应监测（MRM）直接进行完整蛋白质定量已得到证实（J. Am. Soc. Mass Spectrom. 27, 886-896 (2016)）。尽管已知 QqQ-MS 为定量分析提供了非凡的检测灵敏度，但我们发现，在零碰撞能量（ZCE）存在的情况下，完整蛋白质从第一四极杆到第三四极杆质量分析仪的离子传输率不到 5%。为了提高完整蛋白质定量灵敏度，我们研究了离子散射效应、质子转移效应和质量滤波器分辨率宽度对信号丢失的贡献。与各种碰撞诱导解离（CID）气体（N、He 和 Ar）一起分析了肌红蛋白和泛素等蛋白质标准品以及小分子利血平和万古霉素，并在不同气体压力下进行了分析。质量分辨率设置在减少离子传输信号方面起着重要作用。通过将每个侧的质量分辨率窗口缩小 0.35 m/z，可以损失大约 75%-90%的离子信号。多电荷蛋白质经历了额外的质子转移效应，对应于信号降低 10 倍。还使用各种 MRM 求和技术研究了该方法的灵敏度提高。尽管增强程度取决于分析物，但使用多个 MRM 跃迁的求和，观察到泛素的灵敏度提高了 17 倍。用蛋白质对生物基质（人尿和马血浆）进行了加标，以证明该方法的特异性。这项研究为优化 QqQ-MS 用于完整蛋白质定量的使用和灵敏度提供了更多的见解。
