Automated Microvolume Secretome Proteomics Enables Sensitive and Deep Profiling and Noninvasive Single-Embryo Analysis.

Cells continuously deliver proteins into the extracellular space, forming the secretome, which provides a dynamic, biologically informative, and noninvasive readout of cellular functional states. However, conventional secretome proteomics typically requires large sample volumes greater than 2 mL, delivers limited proteome depth, and supports low processing throughput of less than 30 samples a day. Here, we developed an automated microvolume secretome profiling workflow that integrates optimized sample pretreatment and magnetic bead-based proteome sample preparation. This workflow enables efficient secretome profiling from less than 20 μL of conditioned medium, achieving deep proteome coverage of over 3000 proteins with a sample processing throughput exceeding 96 samples per day. Using this workflow, we achieved high-depth, time-resolved secretome profiling from microscale culture medium, capturing temporal changes of secretome. We further applied the method to single mouse embryo culture medium and consistently identified more than 200 secretome proteins per embryo. Notably, Sdc4 and Ooep were consistently observed across developmental stages, highlighting the potential for noninvasive secretome profiling at the single-embryo level. Together, this work establishes a robust and scalable framework for high-depth secretome profiling from ultralow-input samples, broadening the scope of LC-MS-based analysis to microscale and longitudinal biological applications.