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SIMPLEX Enriches Hydrophobic and Lipidated Proteins in Membrane Proteomics Experiments

Year: 2025

Type: article

Abstract: Synaptosomes (Syn) and synaptic junctions (SJ) are key neuronal compartments that have been widely characterized in omics studies to understand neurotransmitter- and signal transduction-related events. While synapses are lipid-rich, multiomics approaches integrating lipids and proteins remain largely underexplored. Liquid-liquid extraction (LLE), commonly used in lipidomics, offers significant potential for multiomics analyses by enabling the extraction of diverse molecular classes from a single sample. However, its impact on protein and phosphoprotein analysis in membrane-enriched samples has not been thoroughly investigated or compared to one-phase extraction methods. In this study, we assessed SIMPLEX (Simultaneous Metabolite, Protein, Lipid Extraction), an LLE-based method, against conventional acetone protein precipitation for mass spectrometry-based protein identification. SIMPLEX proved superior for proteomics and phosphoproteomics of SJ, achieving a 42% enrichment in membrane proteins compared to acetone precipitation. It enriched not only transmembrane proteins but also S-palmitoylated proteins. Enriched phosphoproteins included those with beta-transducin repeats (WD40), Armadillo repeats (ARM), and various transmembrane domains, highlighting the SIMPLEX potential and enhanced performance for multiomics analyses.

Source: PROTEOMICS

DOI: 10.1002/pmic.70016

Authors: Tingting Li, Alexander Wenger, Cristina Coman, Corinna Borutzki, Michael R. Kreutz, Robert Ahrends