Simplified and Efficient Quantification of Low-abundance Proteins at Very High Multiplex via Targeted Mass Spectrometry*
Simplified and Efficient Quantification of Low-abundance Proteins at Very High Multiplex via Targeted Mass Spectrometry*
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DOI：
10.1074/mcp.m113.034660
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发表时间：
2014-04-01
影响因子：
7
通讯作者：
Carr, Steven A.
中科院分区：
文献类型：
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作者：
Burgess, Michael W.;Keshishian, Hasmik;Carr, Steven A.
去除丰富蛋白质并在多肽水平上分成六到八个组分的血浆的LC-MRM-MS是一种被证明是一种定量存在于每毫升低纳克水平的蛋白质的方法。分数-MRM的一个缺点是由于每个生物样品产生多个分数而增加了分析时间。我们现在报告，使用加热的、长的、熔融的硅胶色谱柱(>；30厘米)，填充1.9微米的填料，可以减少或消除LC-MRM-MS之前的分级需求，而不会显著降低相对于分级-MRM的灵敏度或精密度。我们经验性地确定了此类分析的最佳柱长、温度、梯度持续时间和进样量，并利用这些条件研究了检测灵敏度和分析精密度。除了峰容量增加外，填充较小珠子的较长色谱柱可以容忍分析物负荷增加4-6倍，而不会损失稳定性或重现性。与用3微米材料填充的标准12厘米色谱柱相比，较长的色谱柱还提供了4倍的中位数定量限值，并提高了分析精密度。总体而言，优化的层析提供了大约3倍的分析吞吐量，并具有极好的稳健性，定量灵敏度与组分-MRM相比降低了不到2倍。该系统增加多路复用的价值通过配置800多路MRM-MS分析的能力得到证明，该分析在单个分析中运行，包括2400个转变和保留时间计划，以监测400个未标记和重标记的多肽对。
Liquid chromatography-multiple reaction monitoring mass spectrometry (LC-MRM-MS) of plasma that has been depleted of abundant proteins and fractionated at the peptide level into six to eight fractions is a proven method for quantifying proteins present at low nanogram-per-milliliter levels. A drawback of fraction-MRM is the increased analysis time due to the generation of multiple fractions per biological sample. We now report that the use of heated, long, fused silica columns (> 30 cm) packed with 1.9 mu m of packing material can reduce or eliminate the need for fractionation prior to LC-MRM-MS without a significant loss of sensitivity or precision relative to fraction-MRM. We empirically determined the optimal column length, temperature, gradient duration, and sample load for such assays and used these conditions to study detection sensitivity and assay precision. In addition to increased peak capacity, longer columns packed with smaller beads tolerated a 4- to 6-fold increase in analyte load without a loss of robustness or reproducibility. The longer columns also provided a 4-fold improvement in median limit-of-quantitation values with increased assay precision relative to the standard 12 cm columns packed with 3 mu m material. Overall, the optimized chromatography provided an approximately 3-fold increase in analysis throughput with excellent robustness and less than a 2-fold reduction in quantitative sensitivity relative to fraction-MRM. The value of the system for increased multiplexing was demonstrated by the ability to configure an 800-plex MRM-MS assay, run in a single analysis, comprising 2400 transitions with retention time scheduling to monitor 400 unlabeled and heavy labeled peptide pairs.