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.

中文翻译：

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主动仪器参与与实时数据库搜索相结合，可提高样本多路复用工作流程的性能。

已经建立了使用同量异位试剂的定量蛋白质组学作为生物学发现的有力工具。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性和精确度。这种方法的结果是大大降低了光谱采集速率，这需要使用额外的仪器时间来获得全面的蛋白质组学深度。这项工作评估了定量多路复用工作流程中在线和实时光谱识别的性能和优势。实施了实时搜索（RTS）算法，可在几毫秒内识别片段谱，因为它们是使用概率得分获取的，并且仅在确定肽段后才触发定量谱。使用复杂的两个蛋白质组干扰模型和针对激酶的丰度分布图进行有针对性的10重比较，以标准工作流程为基准对RTS-MS3进行了基准测试。应用RTS-MS3方法可在不到50％的采集时间内对10重蛋白质组进行全面表征。这些数据表明，RTS-MS3方法为定量多路复用实验提供了显着的性能改进。