气压驱动的自动化蛋白质组学样品前处理 平台的开发

质谱仪器的快速发展使得基于质谱技术的蛋白质组学研究已经成为蛋白质
高通量定性和定量研究的重要方面。通过质谱鉴定蛋白质需要复杂的样品处理
过程，包括蛋白质提取、还原、烷基化、胰蛋白酶酶解和多肽除盐等过程。复
杂蛋白质的样品处理不仅步骤繁琐且耗时较长； 尤其当样品量较多时，手动处
理样品容易造成样品丢失、样品污染等问题。
本论文设计并制作了一种可以作为蛋白质反应器的微流控器件，并以此微
流控器件为基础开发出气压驱动的自动化蛋白质组学样品前处理平台，实现蛋
白质样品处理的全自动化运行。自主设计的蛋白质反应器成本低廉，降低了样
品处理成本；在气压驱动的自动化样品处理过程中，完成整个样品处理过程只
需约 2 小时，包括 1 小时的酶解时间。使用该自动化平台处理 10 微克 HEK 293T
细胞样品，在 1.4 小时质谱分析中能够鉴定到 4 758 个蛋白；在处理 20 微克
HEK 293T 细胞样品并进行高 pH 值反相分级的 5 个分级中，使用 7 个小时的质
谱分析可以鉴定到 7 557 个蛋白；并且该自动化样品处理平台具有很好的重复
性（单针 HEK 293T 细胞样品 Pearson 相关系数> 0.99，分级 HEK 293T 细胞样
品 Pearson 相关系数> 0.98），且可以用于最低 2 纳克蛋白量的样品处理。在自
动化样品前处理和深度蛋白质组学分析中， 共鉴定出 212 个蛋白激酶，其中包
括 8 种受体酪氨酸激酶。 总之， 该自动化样品前处理平台在蛋白质批量处理和
临床蛋白质组学的应用方面都具有很好的潜力。
 

The rapid development of mass spectrometry instruments has enabled MS-based
proteomics research to become an important aspect of high-throughput identification
and quantification of proteins. There are multiple processing steps in sample
preparation for protein quantification by mass spectrometry, including protein
preconcentration, reduction, alkylation, digestion, and desalting. It is tedious and
time-consuming to process these procedures of numerous complex biological samples,
especially there are large amounts of sample, manual processing of the sample is
likely to cause problems such as sample loss and contamination.
We have built a Pressure-based Auto-Proteome System that enables fully
automated proteomic sample preparation based on pneumatic actuation. The protein
sample preparation step is highly integrated by using a proteomic reactor based on a
microfluidic device of mixed-mode exchange resin packing and C18 membrane. The
self-designed protein reactor is cheap and reduces the cost of sample processing. The
protein samples and reagents are placed in the sample tray of the autosampler. All
protein sample preparation steps are automatically operated by the autosampler and
performed on the microfluidic reactor. It takes only about 2 hours for the sample
preparation, including 1 hours for digestion. Proteomic analysis of 10 μg HEK 293T
cell lysates readily identified 4 758 proteins within 1.4 h LC-MS gradient time, while
7 557 proteins were identified when operate 20 μg HEK 293T cell lysates by fivestep high-pH RP processed and analyzed within only 7 h of LC-MS gradient time.
More importantly, the Auto-Proteome System has good reproducibility (single-shot
analysis Pearson correlation coefficient > 0.99, deep proteome profiling with fivestep high-pH RP fractionation Pearson correlation coefficient > 0.98). The limit of
detection of the Auto-Proteome System was shown to be lower than 2 ng of BSA
protein. Furthermore, the deep proteome profiling it can analysis 212 kinase proteins,
and 8 RTK protein. Overall this proves that the instrument is viable in the pretreatment of proteins. We expect that the Auto-Proteome System can become a
generally applicable sample preparation technology for clinical proteome profiling.
 

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