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首页> 美国卫生研究院文献>Journal of Biomolecular Techniques : JBT >Approaching 100 LC-MS Uptime for Peptide Analyses using Matched Chip Columns
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Approaching 100 LC-MS Uptime for Peptide Analyses using Matched Chip Columns

机译：使用匹配的芯片色谱柱实现100％的LC-MS多肽分析正常运行时间

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>RP-40Overview: High duty cycle nanoLC-MS analyses can be achieved using two columns that alternate between on-line MS analysis and off-line washing and re-equilibration. Using columns with closely matching performance, peptide separations with near identical retention times can be achieved with up to 2X improvement in throughput. Introduction: Inter-column reproducibility is difficult to achieve for capillary columns that operate at nanoliter flow rates. Variations in column ID, inlet- and outlet- frits, column lengths and packing methods, contribute to variations in performance between columns. Chip columns are fabricated to very tight tolerances and as a result achieve closely matched performance. In addition using a recently developed connection system to these chips, variations due to poor connections are eliminated. Near identical performance between columns creates the possibility for increasing throughput in proteomic experiments by alternating between multiple columns during the analysis. In this poster we will be reporting on a microfluidic based method to maximize throughput in LC-MS peptide analyses by alternating between two chip columns. Materials/Equipment: NanoLC-Ultra nanoLC system with cHiPLC nanoflex (Eksigent Technologies, Dublin, CA) in direct injection mode. 15 cm × 75 μm ID ChromXP C18 3μm cHiPLC column (Eksigent Technologies) LTQ iontrap MS (Thermo Scientific, San José, CA) with New Objective (Woburn, MA) nanospray source. Bovine serum albumin reduced/alkylated and digested with trypsin. Summary of Results: We will summarize the inter-run reproducibility of peptide retention times and peak shapes using a dual chip based column set-up over long periods of time and multiple columns. Conclusions: A chip LC platform enables the acquisition of high quality, highly reproducible data. The increased sample throughput obtained using alternating chip based columns and a dual gradient nanoLC pump makes the described system very attractive for studies with larges amounts of samples while at the same time conserving expensive MS analysis time.
机译：> RP-40 概述：可以使用两根在在线MS分析与离线洗涤和重新平衡之间交替的色谱柱来实现高占空比的nanoLC-MS分析。使用性能紧密匹配的色谱柱，可以实现保留时间几乎相同的肽分离，并将通量提高2倍。简介：对于以纳升流速运行的毛细管柱，很难实现柱间重现性。色谱柱ID，入口和出口玻璃料，色谱柱长度和填充方法的变化会导致色谱柱之间性能的变化。芯片柱的制造公差非常严格，因此可以实现紧密匹配的性能。除了将最新开发的连接系统用于这些芯片之外，还消除了由于连接不良而引起的变化。色谱柱之间的性能几乎相同，可以通过在分析过程中在多个色谱柱之间交替来增加蛋白质组学实验的通量。在本海报中，我们将报告一种基于微流体的方法，该方法通过在两个芯片柱之间交替来最大化LC-MS肽分析的通量。材料/设备：具有直接注射模式的cHiPLC nanoflex的NanoLC-Ultra nanoLC系统（Eksigent Technologies，都柏林，加利福尼亚）。 15 cm×75μm内径的ChromXP C183μmcHiPLC色谱柱（Eksigent Technologies）LTQ离子阱MS（Thermo Scientific，SanJosé，CA）和New Objective（Woburn，MA）纳喷雾源。牛血清白蛋白减少/烷基化并用胰蛋白酶消化。结果摘要：我们将使用基于双芯片的色谱柱设置，在较长的时间段内和多根色谱柱上，总结肽保留时间和峰形的批间重现性。结论：芯片LC平台可以采集高质量，高度可重复的数据。使用基于交替芯片的色谱柱和双梯度nanoLC泵获得的增加的样品通量，使所描述的系统非常适合于大量样品的研究，同时节省了昂贵的质谱分析时间。

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