Deep Profiling of Oocyte Aging Enabled by Simple One-Step Vial-Based Pretreatment and Single-Cell Proteomics

Single-cell proteomics is a pivotal technology for studying cellular phenotypes, offering unparalleled insights into cellular heterogeneity and dynamic functions. Technical improvement in mass spectrometry instruments and sample preparation has made proteomics profiling of single mouse oocytes or early embryos feasible in recent years. Yet, developing a simple and robust sample preparation method to enable deep proteomics profiling of single germline cells remains a significant challenge. Herein, we developed a simple one-step vial-based pretreatment (SOViP) for deep label-free single-cell proteomics of germline cells. SOViP integrates all sample preparation procedures into a single step in autosampler vials, yet it is highly efficient and high-throughput in comparison to reported multistep methods. SOViP can be finished within ∼2 h with hands-on time limited to merely a few minutes. On average, over 6500 protein groups can be quantified from a single mouse oocyte using SOViP. In total, 6983 protein groups were identified from single mouse oocytes across an entire reproductive lifespan, offering a valuable proteomics resource for oocyte aging. Unique molecular characteristics of oocytes at different ages were revealed, and a classifier consisting of ten proteins demonstrated accurate age-group classification and fertility-level prediction. Although demonstrated using mouse oocytes in this study, SOViP is adaptable to rare cell samples and other large cells including follicles and preimplantation embryo cells, among others.