Integrated Multi-Omics Enabled by Sequential Extraction for Comprehensive Molecular Profiling of Small Extracellular...

Area of Science:

• Biochemistry
• Molecular Biology
• Biotechnology

Background:

• Small extracellular vesicles (sEVs) contain molecular cargo reflecting their cell of origin.
• sEVs are promising for biomarker discovery and therapeutics.
• Comprehensive characterization of sEVs is hindered by limited sample material.

Purpose of the Study:

• To develop an integrated mass spectrometry-based multi-omics platform for simultaneous protein, lipid, and metabolite analysis from a single sEV sample.
• To maximize sample utilization and enhance molecular coverage and analytical depth.
• To address the challenge of low-input sEV analysis.

Main Methods:

• Integrated mass spectrometry-based multi-omics platform using sequential extraction.
• Iterative tandem mass spectrometry for small-molecule fragmentation.
• Nano-flow proteomics with data-independent acquisition.
• Analysis of sEVs isolated by ultracentrifugation, size-exclusion chromatography, and polymer precipitation.

Main Results:

• Achieved deep and reproducible multi-omic characterization of proteins, lipids, and metabolites from 10 million sEVs.
• Demonstrated platform compatibility with sEVs isolated using various methods.
• Revealed purification-dependent differences in molecular profiles, highlighting yield-purity tradeoffs.

Conclusions:

• The developed platform enables integrated multi-omics analysis from limited sEV samples.
• This approach overcomes a key challenge in low-input sEV analysis.
• Establishes a robust foundation for synergistic biomarker discovery and therapeutic applications of sEVs.