In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells

Omics analysis at single-cell resolution has helped to demonstrate the shaping of cellular heterogeneity on the basis of the expression of various molecules. However, in-depth proteomic analysis of low-quantity samples has remained challenging because of difficulties associated with the measurement of large numbers of proteins by shotgun proteomics using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS). To meet such a demand, we developed a method called in-line sample preparation for efficient cellular proteomics (ISPEC) in which cells were captured, directly lysed, and digested with immobilized trypsin within fused-silica capillaries. ISPEC minimized sample loss during the sample preparation processes with a relatively small number of mammalian cells (<1000 cells) and improved the stability and efficiency of digestion by immobilized trypsin, compared to a conventional preparation method. Using our optimized ISPEC method with nano-LC/MS/MS analysis, we identified 1351, 351, and 60 proteins from 100 cells, 10 cells, and single cells, respectively. The linear response of the signal intensity of each peptide to the introduced cell number indicates the quantitative recovery of the proteome from a very small number of cells. Thus, our ISPEC strategy facilitates quantitative proteomic analysis of small cell populations.单细胞分辨率组学分析（single-cell resolution omics analysis）已助力阐明基于各类分子表达的细胞异质性形成机制。然而，低样本量样品的深度蛋白质组学分析仍面临挑战：采用纳流液相色谱串联质谱（nano-LC/MS/MS）的鸟枪法蛋白质组学（shotgun proteomics）在大规模蛋白质定量检测中存在诸多技术难点。为满足此类研究需求，我们开发了一种高效细胞蛋白质组学在线样品制备法（in-line sample preparation for efficient cellular proteomics，简称ISPEC），该技术可在熔融石英毛细管（fused-silica capillaries）内完成细胞捕获、直接裂解以及固定化胰蛋白酶（immobilized trypsin）酶解。相较于传统样品制备方法，ISPEC可在仅使用<1000个哺乳动物细胞的条件下，大幅降低样品制备过程中的损失，并提升固定化胰蛋白酶酶解的稳定性与效率。将优化后的ISPEC方法与纳流液相色谱串联质谱分析结合，我们分别从100个细胞、10个细胞以及单个细胞中鉴定出1351、351和60种蛋白质。各肽段信号强度与接种细胞数之间呈现线性响应关系，证明该方法可从极少量细胞中实现蛋白质组的定量回收。综上，本ISPEC策略可有效支持小细胞群体的定量蛋白质组学分析。