Laboratory for MEMS Applications, IMTEK, University of Freiburg, Georges-Koehler-Allee 103, 79110 Freiburg, Germany.
Lab Chip. 2020 Aug 21;20(16):2937-2946. doi: 10.1039/d0lc00530d. Epub 2020 Jul 10.
Mass spectrometry has become an important analytical tool for protein research studies to identify, characterise and quantify proteins with unmatched sensitivity in a highly parallel manner. When transferred into clinical routine, the cumbersome and error-prone sample preparation workflows present a major bottleneck. In this work, we demonstrate tryptic digestion of human serum that is fully automated by centrifugal microfluidics. The automated workflow comprises denaturation, digestion and acidification. The input sample volume is 1.3 μl only. A triplicate of human serum was digested with the developed microfluidic chip as well as with a manual reference workflow on three consecutive days to assess the performance of our system. After desalting and liquid chromatography tandem mass spectrometry, a total of 604 proteins were identified in the samples digested with the microfluidic chip and 602 proteins with the reference workflow. Protein quantitation was performed using the Hi3 method, yielding a 7.6% lower median intensity CV for automatically digested samples compared to samples digested with the reference workflow. Additionally, 17% more proteins were quantitated with less than 30% CV in the samples from the microfluidic chip, compared to the manual control samples. This improvement can be attributed to the accurate liquid metering with all volume CVs below 1.5% on the microfluidic chip. The presented automation solution is attractive for laboratories in need of robust automation of sample preparation from small volumes as well as for labs with a low or medium throughput that does not allow for large investments in robotic systems.
质谱分析已成为蛋白质研究的重要分析工具，它以无与伦比的灵敏度，高度平行地鉴定、描述和定量蛋白质。但当它被应用于临床常规时，繁琐易错的样本制备工作流程就成了一个主要瓶颈。在这项工作中，我们演示了使用离心微流控技术全自动进行人血清的胰蛋白酶消化。自动化工作流程包括变性、消化和酸化。输入样本体积仅为 1.3μl。我们用开发的微流控芯片以及手动参考工作流程连续三天对三份人血清进行消化，以评估我们系统的性能。在脱盐和液相色谱串联质谱分析后，在使用微流控芯片消化的样本中总共鉴定出 604 种蛋白质，在使用参考工作流程消化的样本中鉴定出 602 种蛋白质。使用 Hi3 方法进行蛋白质定量，与使用参考工作流程消化的样本相比，自动消化样本的中位数强度 CV 低 7.6%。此外，与手动控制样本相比，在使用微流控芯片消化的样本中，有 17%的蛋白质定量结果 CV 小于 30%，这一改进归因于微流控芯片上的准确液体计量，所有体积 CV 均低于 1.5%。对于需要从小体积样本中实现稳健自动化样本制备的实验室，以及通量较低或中等、无法在机器人系统上进行大量投资的实验室来说，这种自动化解决方案具有吸引力。
