Johnson Alex N T, Huang Jingjing, Marishta Argit, Cruz Edward R, Mariossi Andrea, Barshop William D, Canterbury Jesse D, Melani Rafael, Bergen David, Zabrouskov Vlad, Levine Michael S, Wieschaus Eric, McAlister Graeme C, Wühr Martin
Department of Chemical and Biological Engineering, Princeton University, Princeton, New Jersey, United States; Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey, United States.
Thermo Fisher Scientific, San Jose, California, United States.
Mol Cell Proteomics. 2025 Feb;24(2):100899. doi: 10.1016/j.mcpro.2024.100899. Epub 2024 Dec 24.
Multiplexed proteomics has become a powerful tool for investigating biological systems. Using balancer-peptide conjugates (e.g., TMTproC complementary ions) in the MS2 spectra for quantification circumvents the ratio distortion problem inherent in multiplexed proteomics. However, TMTproC quantification scans require long Orbitrap transients and extended ion injection times to achieve sufficient ion statistics and spectral resolution. Real-time search (RTS) algorithms have demonstrated increased speed and sensitivity by selectively informing precursor peak quantification. Here, we combine complementary ion quantification with RTS (TMTproC-RTS) to enhance sensitivity while maintaining accuracy and precision in quantitative proteomics at the MS2 level. We demonstrate the utility of this method by quantifying protein dynamics during the embryonic development of Drosophila melanogaster (fly), Ciona robusta (sea squirt), and Xenopus laevis (frog). We quantify 7.8k, 8.6k, and 12.7k proteins in each organism, which is an improvement of 12%, 13%, and 14%, respectively, compared with naive TMTproC analysis. For all three organisms, the newly acquired data outperform previously published datasets and provide a diverse, deep, and accurate database of protein dynamics during embryogenesis, which will advance the study of evolutionary comparison in early embryogenesis.
多重蛋白质组学已成为研究生物系统的强大工具。在MS2光谱中使用平衡肽缀合物（例如TMTproC互补离子）进行定量，可规避多重蛋白质组学中固有的比率失真问题。然而，TMTproC定量扫描需要较长的轨道阱瞬态时间和延长的离子注入时间，以实现足够的离子统计和光谱分辨率。实时搜索（RTS）算法已通过选择性地为前体峰定量提供信息，展现出更高的速度和灵敏度。在此，我们将互补离子定量与RTS（TMTproC-RTS）相结合，以提高灵敏度，同时在MS2水平的定量蛋白质组学中保持准确性和精密度。我们通过量化黑腹果蝇（果蝇）、强壮海鞘（海鞘）和非洲爪蟾（青蛙）胚胎发育过程中的蛋白质动态变化，证明了该方法的实用性。我们在每种生物中分别量化了7800、8600和12700种蛋白质，与单纯的TMTproC分析相比，分别提高了12%、13%和14%。对于所有这三种生物，新获得的数据优于先前发表的数据集，并提供了一个关于胚胎发生过程中蛋白质动态变化的多样、深入且准确的数据库，这将推动早期胚胎发生中进化比较的研究。
