Deng Jingren, Lazar Iulia M
Department of Biological Sciences, Virginia Tech, 1981 Kraft Drive, Blacksburg, VA, 24061, USA.
J Am Soc Mass Spectrom. 2016 Apr;27(4):686-98. doi: 10.1007/s13361-015-1332-6. Epub 2016 Feb 16.
The characterization of phosphorylation state(s) of a protein is best accomplished by using isolated or enriched phosphoprotein samples or their corresponding phosphopeptides. The process is typically time-consuming as, often, a combination of analytical approaches must be used. To facilitate throughput in the study of phosphoproteins, a microreactor that enables a novel strategy for performing fast proteolytic digestion and selective phosphopeptide enrichment was developed. The microreactor was fabricated using 100 μm i.d. fused-silica capillaries packed with 1-2 mm beds of C18 and/or TiO2 particles. Proteolytic digestion-only, phosphopeptide enrichment-only, and sequential proteolytic digestion/phosphopeptide enrichment microreactors were developed and tested with standard protein mixtures. The protein samples were adsorbed on the C18 particles, quickly digested with a proteolytic enzyme infused over the adsorbed proteins, and further eluted onto the TiO2 microreactor for enrichment in phosphopeptides. A number of parameters were optimized to speed up the digestion and enrichments processes, including microreactor dimensions, sample concentrations, digestion time, flow rates, buffer compositions, and pH. The effective time for the steps of proteolytic digestion and enrichment was less than 5 min. For simple samples, such as standard protein mixtures, this approach provided equivalent or better results than conventional bench-top methods, in terms of both enzymatic digestion and selectivity. Analysis times and reagent costs were reduced ~10- to 15-fold. Preliminary analysis of cell extracts and recombinant proteins indicated the feasibility of integration of these microreactors in more advanced workflows amenable for handling real-world biological samples. Graphical Abstract ᅟ.
蛋白质磷酸化状态的表征最好通过使用分离或富集的磷酸化蛋白质样品或其相应的磷酸肽来完成。这个过程通常很耗时，因为往往必须结合多种分析方法。为了提高磷酸化蛋白质研究的通量，开发了一种微反应器，它能够实现一种用于快速蛋白水解消化和选择性磷酸肽富集的新策略。该微反应器是用内径100μm的熔融石英毛细管制造的，其中填充了1 - 2mm床层的C18和/或TiO2颗粒。开发了仅用于蛋白水解消化、仅用于磷酸肽富集以及连续蛋白水解消化/磷酸肽富集的微反应器，并用标准蛋白质混合物进行了测试。蛋白质样品吸附在C18颗粒上，用注入到吸附蛋白质上的蛋白水解酶快速消化，然后进一步洗脱到TiO2微反应器上以富集磷酸肽。优化了许多参数以加速消化和富集过程，包括微反应器尺寸、样品浓度、消化时间、流速、缓冲液组成和pH值。蛋白水解消化和富集步骤的有效时间少于5分钟。对于简单样品，如标准蛋白质混合物，就酶促消化和选择性而言，这种方法比传统的台式方法提供了相当或更好的结果。分析时间和试剂成本降低了约10至15倍。对细胞提取物和重组蛋白的初步分析表明，将这些微反应器整合到更适合处理实际生物样品的更先进工作流程中是可行的。图形摘要ᅟ。
