Automated High-Throughput Proteomics Sample Preparation Platform Using DNA Extraction Plate for Industrial Microorganisms

Comprehensive proteomic analyses are indispensable for elucidating protein regulatory networks in microbial chassis cells and accelerating the advancement of cellular factory designs and systems biology models. However, conventional methodologies for generating industrial microbial proteomic data sets remain heavily reliant on labor-intensive, manual workflows that are not only time-inefficient and costly but also inherently prone to human error. In this study, we developed an automated high-throughput proteomics sample preparation workflow (AutoDEP) tailored for industrial microorganisms. By integration of a highly efficient sodium dodecyl sulfate (SDS)-based lysis buffer with a commercially available 96-well DNA extraction plate on the Biomek i7 liquid handling system, AutoDEP enables fully automated processing of 96 samples from cell lysis to clean peptide generation. We rigorously benchmarked the performance of AutoDEP, achieving an excellent sample preparation efficiency with a zero missed cleavage rate exceeding 81%. The intraplate coefficient of variation (CV) showed a median below 7%, while interplate variability demonstrated that over 91% of proteins had CVs less than 20%. Additionally, the interday Pearson correlation coefficient (<i>R</i>²) exceeded 0.98, indicating high reproducibility. Notably, AutoDEP effectively handles protein loading amounts ranging from 5 to 200 μg, showcasing its robust applicability. Collectively, this work demonstrates the potential of AutoDEP to accelerate the construction of cell models based on industrial chassis cells. Furthermore, its robust performance suggests promising applications in large-scale studies, including clinical sample analysis.