Quaternary mixtures can also be deconvoluted. In this case, the spectral library containing the four isomers K6, K29, K48, and K63, was used to calculate relative weights (qi) (Figure ). One additional binary mixture, K48/K63, was measured to obtain all of the necessary relative response factors, including the value for 𝑝𝑝 𝐾𝐾48/𝐾𝐾63 (Figure , Figure ). IM-MS data for approximately thirty ternary and quaternary mixtures were collected to measure the error relative to the actual molar fraction (Table ). A measured error of ≤ 5.2% and a single residue error of 4.8% (SRE corresponds to the difference between the actual and experimental molar fractions for a single type of the isomer) strongly suggests that multiple linear regression analysis can be used to quantify linkage types in mixture of di-Ub isomers (Figure , Supporting Information S17). Validation of the IM-based quantitation approach by AQUA analysis. Lastly, we characterized the di-Ub isomers produced by an E3 ligase using our quantitative IM-MS approach. In the presence of the E2 conjugating enzyme UbcH7, the bacterial effector protein NleL, which is an E3 ligase, generates Ub chains primarily composed of K6 and K48 linkages. After purifying the di-Ub fraction of the enzymatic reaction, bottom-up proteomics with isotopically labeled, Ub-derived peptides-a method referred to as AQUA analysis-confirmed the formation and abundance of K6-and K48-linked di-Ub. We then collected the IM-MS spectrum of the di-Ub fraction in the presence of the apo-myoglobin standard and applied multiple linear regression analysis to different charge states. On average, we found that K6-linked di-Ub comprises 75% while K48-linked di-Ub is only 25% of the di-Ub fraction. These data agree with the AQUA analysis (Figure ), demonstrating the robustness of our quantitative IM-MS-based approach.