Laboratory for the Analysis of Medicines, Center for Interdisciplinary Research on Medicines (CIRM), University of Liege, Quartier hopital, Avenue Hippocrate 15, 4000 Liege, Belgium.
Laboratory for the Analysis of Medicines, Center for Interdisciplinary Research on Medicines (CIRM), University of Liege, Quartier hopital, Avenue Hippocrate 15, 4000 Liege, Belgium.
J Chromatogr A. 2020 May 10;1618:460873. doi: 10.1016/j.chroma.2020.460873. Epub 2020 Jan 13.
Capillary electrophoresis tandem mass spectrometry (CE-MS/MS) is an interesting tool for proteomic analysis as the separation principle is orthogonal to liquid chromatography tandem mass spectrometry (LC-MS/MS). The combination of both techniques can bring complementary information to enlarge proteome coverage. In this study, sample preconcentration techniques were investigated in order to improve sample loading and therefore sensitivity. Dynamic pH junction (DPJ) was found to be the most interesting approach by using 200 mM ammonium acetate (NHAc) adjusted to pH 10.0 as sample matrix. The use of DPJ allowed the identification of more peptides and proteins compared to conventional injections. Moreover, the sheath liquid (SL) composition was optimized in order to enhance signal intensity. A nanoflow SL interface (EMASS-II) was compared to the traditional coaxial SL interface (Triple tube) in terms of number of identified and proteins as well as detection sensitivity (peak area and peak height). MS acquisition was performed using both data-dependent acquisition (DDA) and data-independent acquisition (DIA) modes. The results showed that the combined use of these two acquisition modes provided additional information in terms of identification. Moreover, the use of EMASS-II interface allowed the identification of approximately two times more peptides and proteins. Besides, there was an improvement in sensitivity using EMASS-II as peak height and peak area were improved by 4 and 6-fold, respectively, compared to the Triple tube. Altogether, by combining an efficient sample preconcentration method, a nanoflow CE-MS interface and a hybrid ion-mobility qTOF mass spectrometer, a satisfying sequence coverage was obtained by analyzing 1 µg of E. coli proteome digest.
毛细管电泳串联质谱（CE-MS/MS）作为蛋白质组学分析的一种有趣工具，其分离原理与液相色谱串联质谱（LC-MS/MS）正交。这两种技术的结合可以带来互补信息，扩大蛋白质组覆盖范围。在本研究中，研究了样品预浓缩技术，以提高样品加载量，从而提高灵敏度。通过使用 200 mM 乙酸铵（NHAc）调至 pH 10.0 作为样品基质，发现动态 pH 结（DPJ）是最有趣的方法。与传统进样相比，DPJ 的使用可以鉴定出更多的肽和蛋白质。此外，优化了鞘液（SL）组成，以增强信号强度。纳米流 SL 接口（EMASS-II）与传统的同轴 SL 接口（三管）在鉴定的肽和蛋白质数量以及检测灵敏度（峰面积和峰高）方面进行了比较。MS 采集分别采用数据依赖采集（DDA）和数据非依赖采集（DIA）模式。结果表明，这两种采集模式的结合提供了更多的鉴定信息。此外，使用 EMASS-II 接口可以鉴定出大约两倍多的肽和蛋白质。此外，与三管相比，使用 EMASS-II 接口可以提高灵敏度，峰高和峰面积分别提高了 4 倍和 6 倍。总之，通过结合高效的样品预浓缩方法、纳米流 CE-MS 接口和混合离子淌度 qTOF 质谱仪，分析 1 µg 大肠杆菌蛋白质组消化物即可获得令人满意的序列覆盖率。
