Proteomics and Metabolomics Facility, Institute of Biotechnology, Cornell University, 526 Campus Road, Ithaca, NY, 14853, USA.
State Key Laboratory of Animal Nutrition, Institute of Animal Science, Chinese Academy of Agricultural Sciences, 2 West Yuanmingyuan Road, Beijing, 100193, China.
Anal Bioanal Chem. 2021 Jan;413(2):419-429. doi: 10.1007/s00216-020-03009-5. Epub 2020 Oct 25.
Tandem mass tags (TMTs) have increasingly become an attractive technique for global proteomics. However, its effectiveness for multiplexed quantitation by traditional tandem mass spectrometry (MS) suffers from ratio distortion. Synchronous precursor selection (SPS) MS has been widely accepted for improved quantitation accuracy, but concurrently decreased proteome coverage. Recently, a Real-Time Search algorithm has been integrated with the SPS MS pipeline (RTS MS) to provide accurate quantitation and improved depth of coverage. In this mechanistic study of the impact of exposure to hydrogen sulfide (HS) on the respiration of swine, we used TMT-based comparative proteomics of lung tissues from control and HS-treated subjects as a test case to evaluate traditional MS, SPS MS, and RTS MS acquisition methods on both the Orbitrap Fusion and Orbitrap Eclipse platforms. Comparison of the results obtained by the MS with those of SPS MS and RTS MS methods suggests that the MS-driven quantitative strategies provided a more accurate global-scale quantitation; however, only RTS MS provided proteomic coverage that rivaled that of traditional MS analysis. RTS MS not only yields more productive MS spectra than SPS MS but also appears to focus the analysis more effectively on unique peptides. Furthermore, pathway enrichment analyses of the HS-altered proteins demonstrated that an additional apoptosis pathway was discovered exclusively by RTS MS. This finding was verified by RT-qPCR, western blotting, and TUNEL staining experiments. We conclude that RTS MS workflow enables simultaneous improvement of quantitative accuracy and proteome coverage over alternative approaches (MS and SPS MS). Graphical abstract.
串联质量标签（TMT）技术在全球蛋白质组学中越来越受到关注。然而，其在传统串联质谱（MS）中的多重定量效果受到了比率失真的影响。同步前体选择（SPS）MS 已被广泛接受用于提高定量准确性，但同时也降低了蛋白质组的覆盖度。最近，一种实时搜索算法已经与 SPS MS 管道（RTS MS）集成，以提供准确的定量和提高覆盖率。在这项关于硫化氢（HS）暴露对猪呼吸影响的机制研究中，我们使用基于 TMT 的肺组织比较蛋白质组学作为对照和 HS 处理组的测试案例，评估了传统 MS、SPS MS 和 RTS MS 采集方法在 Orbitrap Fusion 和 Orbitrap Eclipse 平台上的性能。对 MS 与 SPS MS 和 RTS MS 方法获得的结果进行比较表明，MS 驱动的定量策略提供了更准确的全局定量；然而，只有 RTS MS 提供的蛋白质组覆盖度可以与传统 MS 分析相媲美。RTS MS 不仅产生比 SPS MS 更多的有生产力的 MS 谱，而且似乎更有效地将分析集中在独特的肽上。此外，对 HS 改变的蛋白质的途径富集分析表明，RTS MS 方法还发现了一条额外的凋亡途径。这一发现通过 RT-qPCR、western blot 和 TUNEL 染色实验得到了验证。我们得出结论，RTS MS 工作流程能够同时提高定量准确性和蛋白质组覆盖度，优于替代方法（MS 和 SPS MS）。
