Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows
Active Instrument Engagement Combined with a Real-Time Database Search for Improved Performance of Sample Multiplexing Workflows
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DOI：
10.1021/acs.jproteome.8b00899
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发表时间：
2019-03-01
影响因子：
4.4
通讯作者：
Gygi, Steven P.
中科院分区：
文献类型：
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作者：
Erickson, Brian K.;Mintseris, Julian;Gygi, Steven P.
采用同量异位试剂的定量蛋白质组学已被确立为生物发现的有力工具。当前的工作流程通常利用专用的定量光谱来提高定量准确度和精确度。这种方法的一个后果是光谱采集速率的急剧降低，这需要使用额外的仪器时间来实现全面的蛋白质组学深度。这项工作评估了在线和实时光谱识别在定量多路复用工作流程中的性能和优势。实施实时搜索（RTS）算法，以在毫秒内识别片段光谱，因为它们是使用概率评分获得的，并仅在确定肽识别后触发定量光谱。RTS-MS 3使用复杂的双蛋白质组干扰模型和激酶丰度谱的靶向10重比较对标准工作流程进行基准测试。应用RTS-MS 3方法提供了一个10重蛋白质组的全面表征，减少了50%的采集时间。这些数据表明，RTS-MS 3方法为定量多重实验提供了显著的性能改进。
Quantitative proteomics employing isobaric reagents has been established as a powerful tool for biological discovery. Current workflows often utilize a dedicated quantitative spectrum to improve quantitative accuracy and precision. A consequence of this approach is a dramatic reduction in the spectral acquisition rate, which necessitates the use of additional instrument time to achieve comprehensive proteomic depth. This work assesses the performance and benefits of online and real-time spectral identification in quantitative multiplexed workflows. A Real-Time Search (RTS) algorithm was implemented to identify fragment spectra within milliseconds as they are acquired using a probabilistic score and to trigger quantitative spectra only upon confident peptide identification. The RTS-MS3 was benchmarked against standard workflows using a complex two-proteome model of interference and a targeted 10-plex comparison of kinase abundance profiles. Applying the RTS-MS3 method provided the comprehensive characterization of a 10-plex proteome in 50% less acquisition time. These data indicate that the RTS-MS3 approach provides dramatic performance improvements for quantitative multiplexed experiments.