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bioRxiv · 10.1101/2023.07.14.548974

Fully automated and integrated proteomics sample preparation platform for high-throughput drug target identification

Wu, Q.·Zheng, J.·Sui, X.·Fu, C.·Cui, X.·Liao, B.·Ji, H.·Luo, Y.·He, A.·Lu, X.·Tan, C. S. H.·Tian, R.
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Abstract

With the increased demand of large-cohort proteomic analysis, fast and reproducible sample preparation has become the critical issue that needs to be solved. Herein, we developed a fully automated and integrated proteomics sample preparation workflow (autoSISPROT), enabling the simultaneous processing of 96 samples in less than 2.5 hours. Benefiting from its 96-channel all-in-tip operation, protein digestion, peptide desalting, and TMT labeling could be achieved in a fully automated manner. The autoSISPROT demonstrated good sample preparation performances, including >94% of digestion efficiency, nearly 100% of alkylation efficiency, >98% of TMT labeling efficiency, and >0.9 of intra- and inter-batch Pearson correlation coefficients. Furthermore, by combining with cellular thermal shift assay-coupled to mass spectrometry (CETSA-MS), the autoSISPROT was able to process and TMT-label 40 samples automatically and accurately identify the known target of methotrexate. Importantly, taking advantage of the data independent acquisition and isothermal CETSA-MS, the autoSISPROT was well applied for identifying known targets and potential off-targets of 20 kinase inhibitors by automatedly processing 87 samples, affording over a 10-fold improvement in throughput when compared to classical CETSA-MS. Collectively, we developed a fully automated and integrated workflow for high-throughput proteomics sample preparation and drug target identification.

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Wu, Q., Zheng, J., Sui, X., Fu, C., Cui, X., Liao, B., Ji, H., Luo, Y., He, A., Lu, X., Tan, C. S. H., Tian, R.. 2023-07-16. Fully automated and integrated proteomics sample preparation platform for high-throughput drug target identification. https://doi.org/10.1101/2023.07.14.548974

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