SIMPLEX Enriches Hydrophobic and Lipidated Proteins in Membrane Proteomics Experiments

Area of Science:

• Neuroscience
• Biochemistry
• Proteomics

Background:

• Synaptosomes and synaptic junctions are crucial neuronal compartments for understanding signaling.
• Multiomics studies integrating lipids and proteins in these lipid-rich structures are underexplored.
• Liquid-liquid extraction (LLE) shows potential for multiomics but its effect on protein analysis in membrane-enriched samples is unclear.

Purpose of the Study:

• To evaluate SIMPLEX, an LLE-based method, against conventional acetone precipitation for mass spectrometry-based protein identification.
• To assess the impact of LLE on protein and phosphoprotein analysis in membrane-enriched synaptic junction samples.
• To compare the efficiency of SIMPLEX and acetone precipitation for multiomics analyses.

Main Methods:

• Assessed SIMPLEX (Simultaneous Metabolite, Protein, Lipid Extraction), an LLE method.
• Compared SIMPLEX to conventional acetone protein precipitation.
• Utilized mass spectrometry for protein and phosphoprotein identification.

Main Results:

• SIMPLEX demonstrated superior performance for proteomics and phosphoproteomics of synaptic junctions.
• SIMPLEX achieved a 42% enrichment in membrane proteins compared to acetone precipitation.
• SIMPLEX enriched transmembrane and S-palmitoylated proteins, as well as phosphoproteins with specific domains (WD40, ARM).

Conclusions:

• SIMPLEX offers enhanced performance for multiomics analyses of synaptic junctions.
• LLE-based SIMPLEX is a valuable tool for enriching membrane proteins and phosphoproteins.
• This method advances the integration of lipid and protein analyses in neuroscience research.