Accurate and Sensitive Multiplexed Proteomics Using the 7-Plex DiLeuC Complementary Ion Strategy

Abstract High-throughput quantitative proteomics across diverse biological conditions, including varying perturbations and temporal changes, is critical for elucidating disease mechanisms and treatment responses. Advances in mass spectrometry and isobaric labeling technologies have enabled powerful multiplexed analyses; however, commonly used methods rely on low-mass reporter ions that are prone to ratio distortion due to peptide co-isolation and co-fragmentation in complex samples. Complementary ion-based quantification offers an alternative approach by leveraging peptide-specific fragment ions generated at the MS2 level, enabling more accurate and interference-resistant measurements. Here, we present a novel 7-plex dimethylated leucine complementary ion (DiLeuC) isobaric labeling strategy for sensitive and accurate multiplexed proteomics. Through systematic optimization of higher-energy collisional dissociation (HCD) and high-field asymmetric waveform ion mobility spectrometry (FAIMS), we significantly improved complementary ion generation efficiency and proteome coverage. The DiLeuC platform demonstrated high quantitative accuracy and precision across both defined-ratio samples and a human–yeast dual-proteome model, effectively quantifying low-abundance species in the presence of a high-abundance background. Overall, DiLeuC provides a robust and sensitive solution for multiplexed proteomics, enabling accurate complementary ion-based quantification at the MS2 level without requiring SPS-MS3 acquisition. Its efficient complementary ion generation and strong resistance to interference make it well suited for complex biological samples and high-throughput proteomic applications.