Department of Cancer Biology and Blais Proteomics Center, Dana-Farber Cancer Institute, Brigham and Women's Hospital, Harvard Medical School, Boston, Massachusettes, United States.
J Proteome Res. 2010 Dec 3;9(12):6242-55. doi: 10.1021/pr1004696. Epub 2010 Oct 27.
Tandem affinity purification (TAP) coupled with mass spectrometry has become the technique of choice for characterization of multicomponent protein complexes. While current TAP protocols routinely provide high yield and specificity for proteins expressed under physiologically relevant conditions, analytical figures of merit required for efficient and in-depth LC-MS analysis remain unresolved. Here we implement a multidimensional chromatography platform, based on two stages of reversed-phase (RP) separation operated at high and low pH, respectively. We compare performance metrics for RP-RP and SCX-RP for the analysis of complex peptide mixtures derived from cell lysate, as well as protein complexes purified via TAP. Our data reveal that RP-RP fractionation outperforms SCX-RP primarily due to increased peak capacity in the first dimension separation. We integrate this system with miniaturized LC assemblies to achieve true online fractionation at low (≤5 nL/min) effluent flow rates. Stable isotope labeling is used to monitor the dynamics of the multicomponent Ku protein complex in response to DNA damage induced by γ radiation.
串联亲和纯化（TAP）与质谱联用已成为描述多组分蛋白质复合物的首选技术。虽然目前的 TAP 方案通常可在生理相关条件下高效且特异地获得蛋白质，但仍未解决高效和深入的 LC-MS 分析所需的分析性能指标。在此，我们构建了一个多维色谱平台，基于分别在高 pH 和低 pH 下操作的两个反相（RP）分离阶段。我们比较了从细胞裂解物中衍生的复杂肽混合物以及通过 TAP 纯化的蛋白质复合物的 RP-RP 和 SCX-RP 分析的性能指标。我们的数据表明，RP-RP 分级主要由于在第一维分离中增加了峰容量而优于 SCX-RP。我们将该系统与微型化 LC 组件集成，以在低（≤5 nL/min）流速下实现真正的在线分级。稳定同位素标记用于监测多组分 Ku 蛋白复合物在 γ 辐射诱导的 DNA 损伤下的动力学。
