Key Laboratory of Separation Science for Analytical Chemistry, Dalian Institute of Chemical Physics, Chinese Academy of Science, National Chromatographic R. and A. Center, 457 Zhongshan Road, Dalian 116023, China.
Analyst. 2016 Aug 2;141(16):4912-8. doi: 10.1039/c6an00388e.
The pseudo isobaric peptide termini labeling (IPTL) method is a remarkable strategy in quantitative proteomics, and has been efficiently applied in biological studies due to its high quantitative accuracy. However, irreproducible precursor ion selection caused by data dependent acquisition and the chromatographic shift caused by isotope effects limit the wide application of this method. Herein, we expand the use of pseudo IPTL to SWATH MS application and develop a novel quantitative strategy, termed SWATH-pseudo-IPTL, by which the relative quantification could be achieved by comparing the "complete" extracted ion chromatogram (XIC) intensity of MS/MS scan instead of a single intensity measurement in DDA-pseudo-IPTL which only reflected the peptide abundances at that given time. The quantitative analysis of various proportions of mixed HeLa samples revealed the strong accuracy and precision of our SWATH-pseudo-IPTL method, both of which were better than that of the DDA-pseudo-IPTL strategy. SWATH-pseudo-IPTL was also applied to the quantitative profiling of the proteome from human hepatocellular carcinoma cell lines with high and low metastatic potential, and most of the differentially expressed proteins were related to tumorigenesis and tumor metastasis, demonstrating the feasibility of this methodology for biological applications.
拟同位肽末端标记（Pseudo IPTL）方法是定量蛋白质组学中的一种重要策略，由于其具有较高的定量准确性，已在生物研究中得到了有效应用。然而，数据依赖采集导致的不可重现的前体离子选择和同位素效应引起的色谱偏移限制了该方法的广泛应用。在此，我们将拟同位肽末端标记（Pseudo IPTL）方法扩展应用于 SWATH MS 分析，并开发了一种新的定量策略，称为 SWATH-拟同位肽末端标记（SWATH-pseudo-IPTL）。通过比较 MS/MS 扫描的“完整”提取离子色谱图（XIC）强度而不是 DDA-拟同位肽末端标记（DDA-pseudo-IPTL）中仅反映特定时间肽丰度的单一强度测量，实现相对定量。对不同比例混合 HeLa 样品的定量分析表明，我们的 SWATH-拟同位肽末端标记（SWATH-pseudo-IPTL）方法具有较强的准确性和精密度，均优于 DDA-拟同位肽末端标记（DDA-pseudo-IPTL）策略。SWATH-拟同位肽末端标记（SWATH-pseudo-IPTL）还应用于高转移潜能和低转移潜能人肝癌细胞系的蛋白质组定量分析，大多数差异表达蛋白与肿瘤发生和转移有关，证明了该方法在生物学应用中的可行性。
