Development and thorough evaluation of a multi-omics sample preparation workflow for comprehensive LC-MS/MS-based...

Area of Science:

• Biochemistry
• Analytical Chemistry
• Biotechnology

Background:

• Untargeted multi-omics approaches require robust sample preparation to minimize heterogeneity and preserve sample integrity.
• Traditional biphasic solvent systems for sample extraction are often multi-step and complex.
• Monophasic (all-in-one) extraction procedures are emerging as a simpler alternative.

Purpose of the Study:

• To develop and compare ten different mono- and biphasic extraction solvent mixtures for multi-omics analysis.
• To evaluate tissue homogenization parameters for human postmortem muscle and liver samples.
• To identify the optimal extraction method for comprehensive metabolomics, lipidomics, and proteomics datasets.

Main Methods:

• Systematic comparison of ten extraction solvent mixtures (monophasic and biphasic).
• Evaluation of four tissue homogenization parameters using bead homogenization.
• Analysis using untargeted liquid chromatography mass spectrometry (LC-MS) for metabolomics, lipidomics, and proteomics.
• Complementary analysis with 1D SDS-PAGE and BCA assay.

Main Results:

• Optimal homogenization involved bead-processing 20 mg tissue with 200 μL Water:Methanol (1:2) using 3 × 30 s pulses.
• Monophasic extraction solvents generally outperformed biphasic systems for multi-omics data comprehensiveness.
• The Methanol:Acetone (9:1) mixture provided the most comprehensive metabolomics, lipidomics, and proteomics datasets.

Conclusions:

• The Methanol:Acetone (9:1) solvent mixture is optimal for comprehensive multi-omics profiling of human postmortem tissues.
• Optimized sample preparation, including homogenization and extraction, is crucial for maximizing data quality in multi-omics research.
• The identified method shows potential for automation, enabling high-throughput analysis and future clinical applications.