Technical considerations for large-scale parallel reaction monitoring analysis
Technical considerations for large-scale parallel reaction monitoring analysis
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DOI：
10.1016/j.jprot.2013.10.029
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发表时间：
2014-04-04
影响因子：
3.3
通讯作者：
Domon, Bruno
中科院分区：
文献类型：
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作者：
Gallien, Sebastien;Bourmaud, Adele;Domon, Bruno
有针对性的方法已经得到蛋白质组学界的认可，以进行定量实验。然而，目前进行此类实验的参考文献依赖于在三重四极杆质谱仪上进行的选择反应监测（SRM）分析，尽管它受到一些限制。首先，低分辨率四极杆质量分析仪没有呈现足够的选择性来区分分析物与生物样品中通常遇到的干扰。其次，在一个单一的实验中监测的肽的数量往往仍然有限。具有快速测序能力的高分辨率/精确质谱仪的引入使得新型定量方法的开发成为可能。更具体地说，新的四极杆轨道阱质谱仪在平行反应监测（PRM）模式下运行显示检测和定量性能相似或优于SRM中获得的，由于高分辨率轨道阱质量分析仪的选择性增加。该仪器的多功能性，其多路选择的前体离子和操作与不同的四极隔离窗口的能力，使大规模的实验，这需要优化的几个采集参数，以保持高性能的设计。它包括的捕获设备的填充时间和紧凑的调度的肽的洗脱时间的调整，理想地调整on-the-fly.Biological significanceThe本研究构成了一个有价值的基线蛋白质组学社区，以更好地控制之间的权衡灵敏度和分析肽的数量在大规模的平行反应监测（PRM）实验上进行的四极轨道阱仪器。标准采集方法需要仔细设置参数，即一方面根据分辨率和复用程度的控制的填充时间，另一方面根据四极选择窗口。这项研究有助于建立大规模PRM实验的采集参数，同时保持足够的灵敏度。此外，探索了预定肽监测窗口的实时校正，以补偿可能的洗脱时间漂移。这种方法支持在PRM分析中使用缩小的监测窗口，这大大增加了单个LC-MS实验中靶向肽的数量。社区广泛应用所提出的方法可能会建立前所未有的规模靶向蛋白质组学，从而满足系统生物学或生物标志物评估研究的迫切需求。本文是特刊的一部分，题为：蛋白质组学能否填补基因组学与表型学之间的差距？(C)2013年由Elsevier B. V.出版
Targeted methods have gained acceptance among proteomics community to perform quantitative experiments. However, the current reference to conduct such experiments relies on selected reaction monitoring (SRM) analyses performed on triple quadrupole mass spectrometers, although it suffers from some limitations. First, the low resolution quadrupole mass analyzers do not present enough selectivity to discriminate the analytes from interferences commonly encountered in biological samples. Second, the number of peptides monitored in one single experiment often remains limited. The introduction of high resolution/accurate mass instruments with fast sequencing capabilities has enabled the development of novel quantitative methods. More specifically, the new quadrupole-orbitrap mass spectrometer operated in parallel reaction monitoring (PRM) mode showed detection and quantification performances similar or better than those obtained in SRM, due to the increased selectivity of the high-resolution orbitrap mass analyzer. The versatility of the instrument, with its ability to multiplex the selection of precursor ions and to operate with varying quadrupole isolation windows, has enabled the design of large-scale experiments, which require the optimization of several acquisition parameters to maintain high performance. It includes the adjustments of the fill time of the trapping device and the tight scheduling of elution times of the peptides, ideally adjusted on-the-fly.Biological significanceThe present study constitutes a valuable baseline for the proteomics community to better control the trade-off between sensitivity and number of analyzed peptides in large-scale parallel reaction monitoring (PRM) experiments performed on a quadrupole-orbitrap instrument. A standard acquisition method requires careful setting of the parameters, namely the fill time in accordance with the control of the resolving power and the degree of multiplexing on one hand, and the quadrupole selection window on the other hand. This study helps in establishing acquisition parameters for large-scale PRM experiments, while maintaining sufficient sensitivity. In addition, the real-time correction of the scheduled peptide monitoring windows, to compensate for possible elution time drift, was explored. This approach supports the use of narrowed monitoring windows in PRM analyses, which greatly scale up the number of peptides targeted in a single LC-MS experiment The broad application of the presented approaches by the community is likely to allow the establishment of an unprecedented scale for targeted proteomics, thus matching the pressing demand of systems biology or biomarker evaluation studies.This article is part of a Special Issue entitled: Can Proteomics Fill the Gap Between Genomics and Phenotypes? (C) 2013 Published by Elsevier B.V.