Single-Cell Proteomics Using the One-Step Droplet-in-Oil Digestion Method Reveals Proteins Important for Male Meiotic Progression

Different from DNA or RNA, proteins cannot be amplified. The performance of single-cell proteomics is limited by sample loss during sample preparation. Here, we present a one-step droplet-in-oil digestion (OSDO) method that involves one-step water-in-oil processing using cyclohexane or <i>n</i>-heptane, which can reduce the sample adsorption loss and sample volume change due to evaporation and increase the sensitivity and stability of single-cell proteomics. The OSDO is demonstrated to be compatible with tandem mass tag (TMT) labeling to improve the throughput. The OSDO, followed by data-independent acquisition (DIA), quantified more than 3700 proteins per cell during meiotic progression from pachytene to metaphase I, with no correlation between protein and mRNA levels. Inhibition of VPS34 and DNMT1, two proteins up-regulated in metaphase I, both affected metaphase I formation. The OSDO is an easy-to-operate method compatible with both subsequent labeled and unlabeled quantification to expand the depth, throughput, and applicability in single-cell proteomics.