With the addition of apo-myoglobin, we sought to apply a linear deconvolution method to measure the amount of each di-Ub in a binary mixture. A few of the di-Ub isomers, e.g., K29 and K48, exhibit bimodal IM distributions, making it difficult to use gaussian fits with different intensities for quantitation. Thus, we chose to use multiple linear regression analysis, which entailed fitting unknown mixtures to a linear combination of spectra corresponding to individual di-Ub isomers using Euclidean metrics. Weights of each component in a binary mixture (𝑤𝑤 𝑖𝑖 , 𝑤𝑤 𝑗𝑗 ) were obtained, allowing us to calculate relative weights 𝑞𝑞 𝑖𝑖 = 𝑤𝑤 𝑖𝑖 /(𝑤𝑤 𝑖𝑖 + 𝑤𝑤 𝑗𝑗 ) or 𝑞𝑞 𝑗𝑗 = 𝑤𝑤 𝑗𝑗 /(𝑤𝑤 𝑖𝑖 + 𝑤𝑤 𝑗𝑗 ) (Scheme 1). Scheme 1. Spectral deconvolution approach using multiple linear regression analysis. The IM spectrum of a binary mixture (black spectrum) is deconvolved via the IM spectra of single isomers (blue and purple spectra), furnishing the normalized weights of single components that are then be used to create a calibration curve for the calculation of molar fraction of each isomer (χi, χj; shown on the right) in a mixture. The parameter pji in the calibration curve relates to ratios of response factors for different isomers (see Eq. 1).