Gallien Sebastien, Bourmaud Adele, Kim Sang Yoon, Domon Bruno
Luxembourg Clinical Proteomics Center (LCP), CRP-Santé, Strassen, Luxembourg.
Luxembourg Clinical Proteomics Center (LCP), CRP-Santé, Strassen, Luxembourg; University of Luxembourg, Luxembourg.
J Proteomics. 2014 Apr 4;100:147-59. doi: 10.1016/j.jprot.2013.10.029. Epub 2013 Nov 4.
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.
The 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?
靶向方法已在蛋白质组学界获得认可，用于进行定量实验。然而，目前进行此类实验的参考方法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，尽管它存在一些局限性。首先，低分辨率的四极杆质量分析器没有足够的选择性来区分分析物与生物样品中常见的干扰物。其次，在单个实验中监测的肽段数量通常仍然有限。具有快速测序能力的高分辨率/精确质量仪器的引入，使得新型定量方法得以发展。更具体地说，以平行反应监测（PRM）模式运行的新型四极杆 - 轨道阱质谱仪显示出与SRM相当或更好的检测和定量性能，这是由于高分辨率轨道阱质量分析器的选择性提高。该仪器的多功能性，包括其能够对前体离子选择进行多重化以及在不同的四极杆隔离窗口下运行的能力，使得大规模实验的设计成为可能，而大规模实验需要优化多个采集参数以保持高性能。这包括调整捕获装置的填充时间以及肽段洗脱时间的紧密安排，理想情况下是实时调整。
本研究为蛋白质组学界在基于四极杆 - 轨道阱仪器进行的大规模平行反应监测（PRM）实验中更好地控制灵敏度与分析肽段数量之间的权衡提供了有价值的基线。一种标准的采集方法需要仔细设置参数，即一方面根据分辨率和多重化程度的控制来设置填充时间，另一方面设置四极杆选择窗口。本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，还探索了对预定肽段监测窗口进行实时校正，以补偿可能的洗脱时间漂移。这种方法支持在PRM分析中使用更窄的监测窗口，从而极大地增加了单个液相色谱 - 质谱实验中靶向肽段的数量。该社区广泛应用所提出的方法可能会为靶向蛋白质组学建立一个前所未有的规模，从而满足系统生物学或生物标志物评估研究的迫切需求。本文是名为：蛋白质组学能否填补基因组学与表型之间的差距？的特刊的一部分。
