Li Tingting, Wenger Alexander, Coman Cristina, Borutzki Corinna, Kreutz Michael R, Ahrends Robert
Department of Analytical Chemistry, Faculty of Chemistry, University of Vienna, Wien, Austria.
Leibniz-Institut für Analytische Wissenschaften - ISAS - e.V., Dortmund, Germany.
Proteomics. 2025 Aug;25(16):28-39. doi: 10.1002/pmic.70016. Epub 2025 Aug 8.
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.
突触体（Syn）和突触连接（SJ）是关键的神经元区室，在组学研究中已被广泛表征，以了解与神经递质和信号转导相关的事件。虽然突触富含脂质，但整合脂质和蛋白质的多组学方法在很大程度上仍未得到充分探索。脂质组学中常用的液-液萃取（LLE）通过能够从单个样品中提取多种分子类别，为多组学分析提供了巨大潜力。然而，其对富含膜的样品中蛋白质和磷酸化蛋白质分析的影响尚未得到彻底研究，也未与单相萃取方法进行比较。在本研究中，我们评估了基于LLE的方法SIMPLEX（同时代谢物、蛋白质、脂质萃取）与传统丙酮蛋白质沉淀法用于基于质谱的蛋白质鉴定的效果。结果表明，SIMPLEX在SJ的蛋白质组学和磷酸蛋白质组学方面表现更优，与丙酮沉淀相比，膜蛋白富集率提高了42%。它不仅富集跨膜蛋白，还富集S-棕榈酰化蛋白。富集的磷酸化蛋白质包括具有β-转导蛋白重复序列（WD40）、犰狳重复序列（ARM）和各种跨膜结构域的蛋白质，突出了SIMPLEX在多组学分析中的潜力和增强的性能。
