Sensitive and accurate proteome profiling of embryogenesis using Real-Time Search and TMTproC quantification

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 Real-Time Search (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 7.6k, 8.6k, and 12.7k proteins in each organism, which is an improvement of 12%, 13%, and 14%, respectively, compared to naive TMTproC analysis. For all three organisms, the newly acquired data outperform previously published datasets and provides 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（Multiplexed proteomics）已成为研究生物系统的有力工具。采用平衡肽偶联物（如TMTproC互补离子）进行二级质谱（MS2）定量，可规避多重蛋白质组学中固有的比例失真问题。然而，TMTproC定量扫描需要较长的Orbitrap瞬态信号时长与更长的离子注入时间，以获得足够的离子统计量与光谱分辨率。实时搜索（Real-Time Search, RTS）算法通过为前体峰定量提供选择性信息支持，已被证实可提升检测速度与灵敏度。本研究将互补离子定量与实时搜索技术相结合（命名为TMTproC-RTS），在维持二级质谱水平定量蛋白质组学的准确性与精密度的同时，提升了检测灵敏度。我们通过定量分析黑腹果蝇（Drosophila melanogaster，即果蝇）、玻璃海鞘（Ciona robusta，即海鞘）与非洲爪蟾（Xenopus laevis，即爪蟾）胚胎发育过程中的蛋白质动态变化，验证了该方法的实用性。在三种生物中，我们分别定量到7600、8600与12700种蛋白质，相较于未经优化的TMTproC分析，鉴定数量分别提升了12%、13%与14%。对于这三种生物而言，新获得的数据均优于已发表的相关数据集，构建了覆盖广泛、深度充足且结果准确的胚胎发生过程蛋白质动态数据库，将推动早期胚胎发生进化比较研究的发展。