Simple One-step Vial-based Pretreatment for Deep Single-cell Proteomics and Its Application to Oocyte Aging

ABSTRACT 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 instrument and sample preparation has made single-cell proteomics feasible in recent years. Yet, developing a simple and robust sample preparation method to enable deep proteomics profiling of single cells remains a significant challenge. Herein, we developed a simple one-step vial-based pretreatment (SOViP) for deep label-free single-cell proteomics. SOViP integrates all sample preparation procedures into a single step in autosampler vials, yet it is highly efficient and high-throughput. SOViP can be finished within ∼2 h, with hands-on time limited to merely a few minutes. We show that on average over 6,500 can be quantified from a single mouse oocyte using SOViP. In total 6,983 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.