Krisp Christoph, Yang Hao, van Soest Remco, Molloy Mark P
From the ‡Australian Proteome Analysis Facility (APAF), Department of Chemistry and Biomolecular Sciences, Macquarie University, 2109, Sydney, Australia;
§Eksigent, part of AB SCIEX, 94065, Redwood City, California.
Mol Cell Proteomics. 2015 Jun;14(6):1708-19. doi: 10.1074/mcp.M114.046425. Epub 2015 Apr 7.
Comprehensive proteomic profiling of biological specimens usually requires multidimensional chromatographic peptide fractionation prior to mass spectrometry. However, this approach can suffer from poor reproducibility because of the lack of standardization and automation of the entire workflow, thus compromising performance of quantitative proteomic investigations. To address these variables we developed an online peptide fractionation system comprising a multiphasic liquid chromatography (LC) chip that integrates reversed phase and strong cation exchange chromatography upstream of the mass spectrometer (MS). We showed superiority of this system for standardizing discovery and targeted proteomic workflows using cancer cell lysates and nondepleted human plasma. Five-step multiphase chip LC MS/MS acquisition showed clear advantages over analyses of unfractionated samples by identifying more peptides, consuming less sample and often improving the lower limits of quantitation, all in highly reproducible, automated, online configuration. We further showed that multiphase chip LC fractionation provided a facile means to detect many N- and C-terminal peptides (including acetylated N terminus) that are challenging to identify in complex tryptic peptide matrices because of less favorable ionization characteristics. Given as much as 95% of peptides were detected in only a single salt fraction from cell lysates we exploited this high reproducibility and coupled it with multiple reaction monitoring on a high-resolution MS instrument (MRM-HR). This approach increased target analyte peak area and improved lower limits of quantitation without negatively influencing variance or bias. Further, we showed a strategy to use multiphase LC chip fractionation LC-MS/MS for ion library generation to integrate with SWATH(TM) data-independent acquisition quantitative workflows. All MS data are available via ProteomeXchange with identifier PXD001464.
对生物样本进行全面的蛋白质组分析通常需要在质谱分析之前进行多维色谱肽段分级分离。然而，由于整个工作流程缺乏标准化和自动化，这种方法的重现性可能较差，从而影响定量蛋白质组学研究的性能。为了解决这些变量问题，我们开发了一种在线肽段分级分离系统，该系统包括一个多相液相色谱（LC）芯片，该芯片在质谱仪（MS）上游集成了反相色谱和强阳离子交换色谱。我们展示了该系统在使用癌细胞裂解物和未耗尽的人血浆标准化发现型和靶向蛋白质组学工作流程方面的优势。五步多相芯片LC MS/MS采集显示出明显优于未分级样本分析的优势，通过识别更多肽段、消耗更少样本并经常提高定量下限，所有这些都是在高度可重现、自动化的在线配置中实现的。我们进一步表明，多相芯片LC分级分离提供了一种简便的方法来检测许多N端和C端肽段（包括乙酰化N端），这些肽段由于电离特性较差，在复杂的胰蛋白酶肽段基质中难以识别。鉴于细胞裂解物中多达95%的肽段仅在单一盐分级中被检测到，我们利用这种高重现性，并将其与高分辨率MS仪器上的多反应监测（MRM-HR）相结合。这种方法增加了目标分析物的峰面积并提高了定量下限，而不会对方差或偏差产生负面影响。此外，我们展示了一种使用多相LC芯片分级分离LC-MS/MS生成离子库以与SWATH（TM）数据非依赖采集定量工作流程集成的策略。所有MS数据可通过ProteomeXchange获得，标识符为PXD001464。
