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A mass defect-based labeling strategy provides high accuracy as an MS 1 -centric quantification method, avoiding the ratio compression that affects isobaric label-based reporter ion quantification. We have developed cost-effective 5-plex mass defect N, N-dimethyl leucine (mdDiLeu) tags for quantification of various biological samples with increased multiplexing at a given resolving power afforded by the addition of mass difference isotopologues. The combination of mass difference and mass defect produces two labeled peak clusters separated by 5 Da in MS 1 spectra that are detected as five isotopic peaks at high resolution with mass differences of 15, 17, and 18 mDa per tag. Synthesis of each of the 5-plex mdDiLeu tags is accomplished by a single straightforward reaction step, making it accessible to any lab. To demonstrate 5-plex mdDiLeu for quantitative proteomics, we perform proof-of-principle experiments of mdDiLeu-labeled Saccharomyces cerevisiae lysate digest on an Orbitrap Fusion Lumos mass spectrometer.

High-Resolution Enabled 5-plex Mass Defect-Based N,N-Dimethyl Leucine Tags for Quantitative Proteomics