Development of a high-throughput proteome sample preparation workflow based on efficient protein extraction to...

Mass spectrometry-based proteomics provides a powerful tool for the rational design of yeast cell factories. However, the rigid yeast cell wall architecture and the fast-growing scale of strain libraries present certain challenges for efficient protein extraction and high-throughput proteomic profiling. Conventional protein extraction methods either demand lengthy incubation periods or specialized equipment, leading to limited sample throughput and low efficiency. Herein, we report an efficient protein extraction strategy using a solvent combination of ionic liquid, guanidine hydrochloride, and lithium acetate under heat treatment (IGL-H). This method enables rapid protein extraction and is compatible with diverse yeast species across different growth phases. Compared with the classical glass bead grinding method, the IGL-H method affords higher protein yield and comparable or superior proteome coverage, and recovers higher proportions of integral membrane proteins of the endomembrane trafficking system. Furthermore, this method is readily amenable to liquid handling workstations for high-throughput proteome sample preparation by using magnetic solid-phase alkylation (mSPA) materials in less than 1.5 h, achieving Pearson correlation coefficients exceeding 0.97. Applying this pipeline to study proteome dynamics of Candida utilis under different nitrogen sources revealed an adaptive strategy involving carbon flux-specific allocation. These results establish the IGL-H extraction strategy as an effective solution for rapid protein extraction and high-throughput proteomic analysis of yeast strains, advancing rational cell factory design in synthetic biology.