Cruz Edward R, Johnson Alex N T, Pujari Vyas, Zhang Qi, Nguyen Thao, Stadlmeier Michael, Wang Jessica, Jacob Cristina C, McAlister Graeme C, Remes Philip M, Wühr Martin
Department of Molecular Biology, Princeton University, Princeton, New Jersey 08544, United States.
Lewis-Sigler Institute for Integrative Genomics, Princeton University, Princeton, New Jersey 08544, United States.
J Proteome Res. 2025 Aug 6. doi: 10.1021/acs.jproteome.5c00356.
Proteomic workflows have traditionally been divided into discovery-based and targeted approaches with instrumentation optimized specifically for each. Discovery experiments typically utilize high-resolution analyzers, while targeted workflows rely on the sensitivity and specificity of triple quadrupole systems. Recently, a quadrupole-ion trap instrument (Stellar MS) has demonstrated superior performance for targeted applications compared to conventional triple quadrupoles. In this study, we expand the capabilities of this platform to multiplexed shotgun proteomics using complement reporter ion quantification in an ion trap (iTMTproC). Benchmarking experiments with defined standards show that iTMTproC achieves quantification accuracy and interference reduction comparable to MultiNotch MS on the Orbitrap Fusion Lumos, a dedicated quadrupole-ion trap-Orbitrap tribrid instrument optimized for this purpose. Notably, iTMTproC quantifies slightly more proteins than does MultiNotch MS. We further validate this approach through a developmental time-series analysis of frog embryos, obtaining proteomic data nearly indistinguishable from those from MultiNotch MS, with slightly increased protein quantification depth. These findings significantly extend the functionality of targeted instrumentation, underscoring the versatility of quadrupole-ion trap systems and providing cost-effective access to highly accurate, multiplexed quantitative shotgun proteomics.
传统上，蛋白质组学工作流程分为基于发现的方法和靶向方法，每种方法都有专门优化的仪器。发现实验通常使用高分辨率分析仪，而靶向工作流程则依赖于三重四极杆系统的灵敏度和特异性。最近，一种四极杆-离子阱仪器（Stellar MS）在靶向应用中表现出优于传统三重四极杆的性能。在本研究中，我们利用离子阱中的互补报告离子定量（iTMTproC），将该平台的功能扩展到多重鸟枪法蛋白质组学。使用定义标准的基准实验表明，iTMTproC在定量准确性和干扰减少方面可与用于此目的而优化的专用四极杆-离子阱-轨道阱三合一仪器Orbitrap Fusion Lumos上的MultiNotch MS相媲美。值得注意的是，iTMTproC定量的蛋白质比MultiNotch MS略多。我们通过对青蛙胚胎的发育时间序列分析进一步验证了这种方法，获得了与MultiNotch MS几乎无法区分的蛋白质组学数据，蛋白质定量深度略有增加。这些发现显著扩展了靶向仪器的功能，强调了四极杆-离子阱系统的多功能性，并提供了经济高效的途径来进行高精度、多重定量鸟枪法蛋白质组学研究。
