Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Anal Chem. 2010 Apr 1;82(7):2784-96. doi: 10.1021/ac902710k.
Many cellular processes are driven by protein complexes. Although the identification of protein components in such complexes has become almost a routine matter, accurate determination of their stoichiometry within a protein complex is still a challenge. We have established a method to determine the stoichiometries of protein complexes using absolute quantification (AQUA) with the help of synthetic standard peptides in combination with multiple reaction monitoring (MRM). Our approach is exemplified by the analysis of the human spliceosomal hPrp19/CDC5L complex, which consists of seven individual proteins and plays a crucial role in the assembly of the fully catalytically active spliceosome during pre-mRNA splicing. We evaluated several conditions for complete hydrolysis of the protein complex and found that the denaturing conditions under which hydrolysis is performed are absolutely crucial for accurately determining protein stoichiometries within this complex. In addition, we tested the suitability of different AQUA peptides and further compared different MS techniques to read out the relative signal intensities that were then used in absolute quantification. Our analyses revealed that dependent on the denaturing conditions different stoichiometries within the complex were obtained. The most consistent results were obtained by enzymatic hydrolysis in the presence of acetonitrile in combination with MRM.
许多细胞过程是由蛋白质复合物驱动的。虽然鉴定这样的复合物中的蛋白质成分几乎已经成为常规事项，但准确确定蛋白质复合物中的化学计量仍然是一个挑战。我们已经建立了一种使用绝对定量 (AQUA) 结合合成标准肽和多重反应监测 (MRM) 来确定蛋白质复合物化学计量的方法。我们的方法通过分析人类剪接体 hPrp19/CDC5L 复合物得到了例证，该复合物由七个独立的蛋白质组成，在 pre-mRNA 剪接过程中组装完全催化活性的剪接体中起着至关重要的作用。我们评估了几种完全水解蛋白质复合物的条件，发现水解过程中的变性条件对于准确确定该复合物中蛋白质的化学计量绝对至关重要。此外，我们还测试了不同 AQUA 肽的适用性，并进一步比较了不同的 MS 技术来读取相对信号强度，然后用于绝对定量。我们的分析表明，取决于变性条件，复合物中获得了不同的化学计量。最一致的结果是在存在乙腈的情况下通过酶促水解并结合 MRM 获得的。
