AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation

Lu, Xue1; Wang, Zhikun1; Gao, Yan2; Chen, Wendong1; Wang, Lingjue1; Huang, Peiwu1; Gao, Weina1; Ke, Mi1; He, An1; Tian, Ruijun1

2020-07-07

10.1021/acs.analchem.0c00752

ANALYTICAL CHEMISTRY   影响因子和分区

0003-2700

1520-6882

With recent advances in LC-MS systems, current MS-based proteomics has an increasing need for automated, high-throughput sample preparation with neglectable sample loss. In this study, we developed a microfluidic system for fully automated proteomics sample preparation. All of the required proteomics sample preparation steps for both protein digestion and peptide fractionation are fully integrated into a disposable plastic chip device (named AutoProteome Chip). The AutoProteome Chip packed with mixed-mode ion exchange beads and C18 membrane in tandem could be fabricated with very low cost and high stability in organic reagents. Benefiting from its low backpressure, the AutoProteome Chip could be precisely driven by gas pressure, which could be easily multiplexed. As low as 2 ng of standard protein BSA could be trapped into the AutoProteome chip and processed within 2 h. Fully automated processing of 10 mu g of protein extracts of HEK 293T cells achieved more than 97% of digestion efficiency with missed cleavage less than 2 and comparable performance with conventional approaches. More than 4700 proteins could be readily identified within 80 min of LC-MS analysis with good label-free quantification performance (Pearson correlation coefficient >0.99). Furthermore, deep proteome profiling by integrated high-pH RP fractionation in the same AutoProteome Chip resulted in more than 7500 proteins being identified from only 20 pg of protein extracts of HEK 293T cells and comparable reprodicibility as single-shot analysis. The AutoProteome Chip system provided a valuable prototype for developing a fully automated proteome analysis workflow and for proteomic applications with high demand for processing throughput, reproducibility, and sensitivity.

SCI ; EI

英语

NI期刊 ; NI论文

第一 ; 通讯

National Natural Science Foundation of China[31700088] ; China State Key Basic Research Program Grants[2016YFA0501403][2016YFA0501404] ; Guangdong Provincial Fund for Distinguished Young Scholars[2019B151502050] ; Guangdong Provincial Natural Science Grant[2016A030312016] ; Shenzhen Innovation of Science and Technology Commission[JCYJ20170412154126026]

Chemistry

Chemistry, Analytical

WOS:000548541200037

AMER CHEMICAL SOC

CHEMISTRY

Web of Science

1.Southern Univ Sci & Technol, Dept Chem, Shenzhen 518055, Peoples R China
2.Hochuen Med Technol Co Ltd, Shenzhen 518109, Peoples R China

Lu, Xue,Wang, Zhikun,Gao, Yan,et al. AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation[J]. ANALYTICAL CHEMISTRY,2020,92(13):8893-8900.

Lu, Xue.,Wang, Zhikun.,Gao, Yan.,Chen, Wendong.,Wang, Lingjue.,...&Tian, Ruijun.(2020).AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation.ANALYTICAL CHEMISTRY,92(13),8893-8900.

Lu, Xue,et al."AutoProteome Chip System for Fully Automated and Integrated Proteomics Sample Preparation and Peptide Fractionation".ANALYTICAL CHEMISTRY 92.13(2020):8893-8900.