Accurate and Sensitive Multiplexed Proteomics Using the 7-Plex DiLeuC Complementary Ion Strategy
Year: 2026
Type: article
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.
Authors: Jingwei Zhang, Zicong Wang, Yajing Lu, Yunxiao Yao, Lingjun Li
Institution: University of Wisconsin–Madison
Language: English
Subfield: Spectroscopy
Field: Chemistry
Domain: Physical Sciences
Open Access status: hybrid
Estimated APC paid: $4,500
Funders: Wisconsin Alumni Research Foundation, University of Wisconsin-Madison, National Institutes of Health, National Institute on Aging, NIH Office of the Director, National Institute of General Medical Sciences, National Institute of Diabetes and Digestive and Kidney Diseases, National Center for Research Resources
Awards: Acquisition of a High-Field Dual Source FTICR-MS for Pharmaceutical Research, G1803960457, G2075862466, Creating a region- specific biomolecular atlas of the brain of Alzheimer’s disease, Mass Spectrometric Studies of Neuropeptides in Feeding, National Center for Quantitative Biology of Complex Systems, DiLeu-enabled multiplexed quantitation for biomarker discovery and validation in Alzheimer’s disease
OpenAlex ID: W7220649263