Department of Applied Genomics, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.
Department of Frontier Research and Development, Kazusa DNA Research Institute, Kisarazu, Chiba, Japan.
Mol Cell Proteomics. 2024 Apr;23(4):100745. doi: 10.1016/j.mcpro.2024.100745. Epub 2024 Mar 4.
In recent years, there has been a growing demand for low-input proteomics, particularly in the context of single-cell proteomics (SCP). In this study, we have developed a lauryl maltose neopentyl glycol (LMNG)-assisted sample preparation (LASP) method. This method effectively reduces protein and peptide loss in samples by incorporating LMNG, a surfactant, into the digestion solution and subsequently removing the LMNG simply via reversed phase solid-phase extraction. The advantage of removing LMNG during sample preparation for general proteomic analysis is the prevention of mass spectrometry (MS) contamination. When we applied the LASP method to the low-input SP3 method and on-bead digestion in coimmunoprecipitation-MS, we observed a significant improvement in the recovery of the digested peptides. Furthermore, we have established a simple and easy sample preparation method for SCP based on the LASP method and identified a median of 1175 proteins from a single HEK239F cell using liquid chromatography (LC)-MS/MS with a throughput of 80 samples per day.
近年来,人们对低投入蛋白质组学的需求不断增长,特别是在单细胞蛋白质组学(SCP)方面。在本研究中,我们开发了一种月桂基麦芽糖新戊二醇(LMNG)辅助的样品制备(LASP)方法。该方法通过将表面活性剂 LMNG 加入消化液中,并通过反相固相萃取简单地去除 LMNG,有效地减少了样品中的蛋白质和肽的损失。在用于常规蛋白质组学分析的样品制备过程中去除 LMNG 的优点是防止质谱(MS)污染。当我们将 LASP 方法应用于低投入 SP3 方法和共免疫沉淀-MS 中的珠上消化时,我们观察到消化肽的回收率有了显著提高。此外,我们还基于 LASP 方法建立了一种简单易用的 SCP 样品制备方法,使用 LC-MS/MS 从单个 HEK239F 细胞中鉴定到中位数为 1175 种蛋白质,每天的通量为 80 个样本。