Departments of Chemistry and Molecular Biosciences, Proteomics Center of Excellence, Northwestern University, Evanston, IL, USA.
Proteomics. 2014 May;14(10):1158-64. doi: 10.1002/pmic.201300381. Epub 2014 Apr 10.
The direct analysis of intact proteins via MS offers compelling advantages in comparison to alternative methods due to the direct and unambiguous identification and characterization of protein sequences it provides. The inability to efficiently analyze proteins in the "middle mass range," defined here as proteins from 30 to 80 kDa, in a robust fashion has limited the adoption of these "top-down" methods. Largely, a result of poor liquid chromatographic performance, the limitations in this mass range may be addressed by alternative separations that replace chromatography. Herein, the short migration times of CZE-ESI-MS/MS have been extended to size-sorted whole proteins in complex mixtures from Pseudomonas aeruginosa PA01. An electrokinetically pumped nanospray interface, a coated capillary, and a stacking method for on-column sample concentration were developed to achieve high-loading capacity and separation resolution. We achieved full width at half maximum of 8-16 s for model proteins up to 29 kDa and identified 30 proteins in the mass range of 30-80 kDa from P. aeruginosa PA01 whole cell lysate. These results suggest that CZE-ESI-MS/MS is capable of identifying proteins in the middle mass range in top-down proteomics.
通过 MS 对完整蛋白质进行直接分析与其他方法相比具有很大的优势，因为它可以直接、明确地鉴定和描述蛋白质序列。由于无法以稳健的方式高效地分析“中等质量范围”（定义为 30 至 80 kDa 的蛋白质）内的蛋白质，因此这些“自上而下”的方法的应用受到了限制。主要是由于较差的液相色谱性能，这种质量范围的限制可以通过替代分离方法来解决，这些方法可以替代色谱法。在此，通过对复杂混合物中的 Pseudomonas aeruginosa PA01 中的大小分级的全蛋白质进行 CE-ESI-MS/MS 的短迁移时间分析，扩展了该方法的应用范围。为了实现高负载能力和分离分辨率，我们开发了电泵纳米喷雾接口、涂层毛细管和柱上样品浓缩的堆积方法。我们实现了模型蛋白质的半峰全宽为 8-16 s，直到 29 kDa，并从 Pseudomonas aeruginosa PA01 全细胞裂解物中鉴定出 30 种 30-80 kDa 质量范围内的蛋白质。这些结果表明，CE-ESI-MS/MS 有能力在自上而下的蛋白质组学中鉴定中等质量范围的蛋白质。
