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对蛋白质或多肽的高效分离,将有利于降低肽段信号之间的干扰,保证规模化的蛋白质组学深度覆盖鉴定,并提高定量蛋白质组分析的准确性。本研究采用一种新型的等电聚焦预分离系统(OFFGEL),考察系统对293T细胞在蛋白质与肽段水平的分级分离效果,在OFFGEL系统中聚焦后的蛋白质或肽段可以从溶液中直接回收,与下游的LC-MS/MS肽段鉴定流程兼容。实验结果表明,肽段的分离效果明显优于蛋白质的分离,分级分离后各馏分对应的等电点位置与肽段的理论等电点分布高度一致,每个馏分中单独鉴定的肽段比例超过90%,显示了该系统对肽段的高分辨分离能力,结合生物质谱技术,在293T细胞中实现6727个蛋白质的规模化鉴定,表明该系统在复杂体系蛋白质组研究中的应用潜力。
The efficient separation of proteins or peptides will help to reduce the interference between peptide signals, ensure large-scale coverage of proteomic deep coverage and improve the accuracy of quantitative proteomic analysis. In this study, a new type of isoelectric focusing pre-separation system (OFFGEL) was used to investigate the fractionation of 293T cells at the protein and peptide levels. The focused proteins or peptides in the OFFGEL system can be recovered directly from the solution , Compatible with downstream LC-MS / MS peptide identification procedures. The experimental results show that the separation efficiency of peptides is better than that of proteins. The isoelectric point of each fraction corresponds well with the theoretical isoelectric point distribution of peptides after fractionation. The proportion of peptides identified in each fraction exceeds 90%, showing the system’s ability of high resolution separation of peptides. In combination with bio-mass spectrometry, the scale-up of 6,727 proteins in 293T cells was demonstrated, indicating the potential of this system in the study of proteomes of complex systems.