Automated strain-to-peptide conversion: a high-throughput proteome analysis platform empowering rational design of...

Mass spectrometry-based proteomics offers a powerful tool for characterizing enzyme expression in engineered strains, yet rapid generation of large strain libraries creates proteomic analysis bottlenecks. The critical limitation lies in manual sample preparation-protein extraction, denaturation, reduction, desalting, and digestion-which is time-consuming and risks compromising reproducibility. To overcome this bottleneck, we developed a novel "strain-to-peptide conversion" (SPC) strategy for high-throughput proteome profiling in microbial cell factories. This automated workflow integrates bacterial lysis, magnetic solid-phase alkylation (mSPA)-based protein enrichment, contaminant removal, and rapid digestion through a commercial liquid handling system, processing 96 samples within 1 hour. Compared to the well-established single-pot solid-phase-enhanced sample preparation (SP3) method, SPC achieves a 94% reduction in processing time while maintaining equivalent protein identification depth. Furthermore, the quantification of membrane proteins was increased by 28%. Meanwhile, the method demonstrated exceptional reproducibility, with intra- and inter-batch Pearson correlation coefficients exceeding 0.95. Leveraging this platform, we processed 96 E. coli samples simultaneously, with reliable quantitative data revealing significant regulation of proteins primarily associated with translation, transmembrane transport, and metabolic processes following overexpression of key tricarboxylic acid (TCA) cycle enzymes. These results establish the SPC strategy as an efficient high-throughput solution for large-scale strain proteome analysis, advancing rational cell factory design in metabolic engineering and synthetic biology.