National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, 116023, China; University of Chinese Academy of Sciences, Beijing, China.
National Chromatographic Research and Analysis Center, Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, Dalian, 116023, China.
Anal Chim Acta. 2018 Feb 13;1000:172-179. doi: 10.1016/j.aca.2017.10.023. Epub 2017 Oct 27.
In recent years, various enzymatic microreactors and on-line enzyme digestion strategies have been widely applied in high throughput proteome analysis. However, the incomplete and irreproducible digestion would introduce some unexpected variations in comparative proteome quantification when the samples are digested and then chemically isotope labeled in different aliquots. To address these problems, we developed an integrated platform for high throughput proteome quantification with combination of on-line low miss-cleavage protein digestion by an ultra-performance immobilized enzymatic reactor, on-line dimethyl labeling onto a C18 precolumn, peptide separation by two-dimensional nano liquid chromatography and MS detection. Compared to traditional off-line method, such a platform exhibits obvious advantages such as high sensitivity, throughput, accuracy, precision and ease of automation. All these results demonstrated that such a platform might become a promising technique for the quantitative proteome analysis.
近年来，各种酶微反应器和在线酶解策略已广泛应用于高通量蛋白质组分析中。然而，当样品在不同等分试样中进行酶解和化学同位素标记时，不完全和不可重复的酶解会在比较蛋白质组定量分析中引入一些意想不到的变化。为了解决这些问题，我们开发了一种集成的高通量蛋白质组定量分析平台，该平台结合了在线低错切蛋白酶解，通过超高效固定酶反应器进行，在线二甲基化标记到 C18 预柱上，通过二维纳升液相色谱和 MS 检测进行肽分离。与传统的离线方法相比，该平台具有灵敏度高、通量高、准确性高、精密度高和易于自动化等明显优势。所有这些结果表明，该平台可能成为定量蛋白质组分析的一种有前途的技术。
