Increased selectivity, analytical precision, and throughput in targeted proteomics.
Increased selectivity, analytical precision, and throughput in targeted proteomics.
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DOI：
10.1074/mcp.m110.002931
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发表时间：
2011-02
期刊：
影响因子：
--
通讯作者：
Domon B
中科院分区：
文献类型：
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作者：
Kiyonami R;Schoen A;Prakash A;Peterman S;Zabrouskov V;Picotti P;Aebersold R;Huhmer A;Domon B
蛋白质组学正在逐渐用假设驱动的定量实验来补充大规模的定性研究。在选定的反应监测模式下，在三重四极杆仪器上进行的目标分析具有高选择性和低检测限的特点；然而，同时分析多个分析物是以牺牲灵敏度为代价的，因为由于几个转变的限制，停留时间和/或选择性减少。提出了一种新的数据采集范式，其中选择的反应监测以两种方式进行，以同时定量和确认目标多肽的身份。在预定的洗脱时间窗口期间连续监测第一组初级转变，以精确地定量每个肽。此外，在数据相关事件中获取一组六到八个转换，当所有主要转换超过预设阈值时触发。这些额外的跃迁被用来产生复合串联质谱图，以正式确认目标多肽的身份。将该技术应用于一种酵母裂解物的胰酶消化分析，以演示该技术的性能。在一次60分钟的实验中，我们展示了检测极限，最低可检测到数十个阿托莫尔，吞吐量超过6000个跃迁。这项技术被整合到一个线性工作流程中，包括实验设计、数据获取和数据评估，使大规模蛋白质组研究成为可能。
Proteomics is gradually complementing large shotgun qualitative studies with hypothesis-driven quantitative experiments. Targeted analyses performed on triple quadrupole instruments in selected reaction monitoring mode are characterized by a high degree of selectivity and low limit of detection; however, the concurrent analysis of multiple analytes occurs at the expense of sensitivity because of reduced dwell time and/or selectivity due to limitation to a few transitions. A new data acquisition paradigm is presented in which selected reaction monitoring is performed in two ways to simultaneously quantify and confirm the identity of the targeted peptides. A first set of primary transitions is continuously monitored during a predetermined elution time window to precisely quantify each peptide. In addition, a set of six to eight transitions is acquired in a data-dependent event, triggered when all the primary transitions exceed a preset threshold. These additional transitions are used to generate composite tandem mass spectra to formally confirm the identity of the targeted peptides. This technique was applied to analyze the tryptic digest of a yeast lysate to demonstrate the performance of the technique. We showed a limit of detection down to tens of attomoles injected and a throughput exceeding 6000 transitions in one 60-min experiment. The technique was integrated into a linear work flow, including experimental design, data acquisition, and data evaluation, enabling large scale proteomic studies.