With the increased demand of large-cohort proteomic analysis, fast and reproducible sample preparation has become a critical issue. Herein, we developed a fully automated proteomics sample preparation workflow (autoSISPROT). This platform enables simultaneous processing of 96 samples in less than 2.5 hours, achieving protein digestion, desalting, and TMT labeling through 96-channel all-in-tip operations. The results demonstrated excellent sample preparation performance with >94% digestion efficiency, >98% TMT labeling efficiency, and >0.9 of intra- and inter-batch Pearson correlation coefficients. Application of autoSISPROT on cellular thermal shift assay (CETSA) allows automatic and high throughput identification of drug targets. Specifically, by automatically processing 87 samples of isothermal shift assay using data independent acquisition and, autoSISPROT identified known targets as well as potential off-targets of 20 kinase inhibitors, affording over a 10-fold improvement in throughput compared to classical CETSA-MS. This fully automated workflow offers a high-throughput solution for proteomics sample preparation and drug target/off-target identification.

This article is Open Access

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