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

Area of Science:

• Proteomics
• Cell Biology
• Biotechnology

Background:

• The secretome offers a dynamic readout of cellular states but conventional proteomics requires large volumes and is low-throughput.
• Limitations include large sample volume requirements (>2 mL), limited proteome depth, and low processing capacity (<30 samples/day).

Purpose of the Study:

• To develop an automated microvolume secretome profiling workflow for high-depth analysis from ultralow-input samples.
• To enhance sample processing throughput and enable time-resolved secretome analysis.

Main Methods:

• Developed an automated workflow integrating optimized sample pretreatment and magnetic bead-based proteome preparation.
• Utilized microvolume (<20 μL) conditioned medium for secretome profiling.
• Applied liquid chromatography-mass spectrometry (LC-MS) for deep proteome coverage.

Main Results:

• Achieved deep proteome coverage (>3000 proteins) from <20 μL of sample.
• Enabled high sample processing throughput (>96 samples/day).
• Successfully profiled secretome from single mouse embryos, identifying >200 proteins per embryo and tracking temporal changes.

Conclusions:

• Established a robust and scalable framework for high-depth secretome profiling from ultralow-input samples.
• Broadened the scope of LC-MS-based analysis to microscale and longitudinal biological applications.
• Demonstrated potential for noninvasive, single-embryo secretome profiling.