Expanding Targeted Instrumentation for Discovery Applications: Complement Reporter Ion Quantification with a Quadrupole–Ion Trap Instrument

Abstract

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 MS³ 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 MS³. We further validate this approach through a developmental time-series analysis of frog embryos, obtaining proteomic data nearly indistinguishable from those from MultiNotch MS³, 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.