IM spectra of seven ubiquitin dimers. Previous studies have shown that even under denaturing conditions, the isomers of di-Ub can still be distinguished by IM-MS. We employed similar conditions to resolve the different isomers. As shown in Use of apo-myoglobin as an internal standard. Quantitative IM-MS is challenging due to the dependence of IM distribution on external conditions and the ESI MS experimental setup. Deviations in gas temperature in the mobility cell as well as small variations in the position and shape of the nanoESI emitter may lead to random drift in the distributions of di-Ub ions. To compensate for these deviations, we introduced an internal calibrant to calibrate collision cross sections (CCS) across di-Ub isomers. We chose apo-myoglobin because its molecular weight and charge distribution are similar to di-Ub and its signal does not overlap with that of di-Ub in the mobility spectra (Figure ). Moreover, the CCS values of apo-myoglobin added as an internal or external standard only deviate by 0.6% from the reference values (Table ). The inclusion of a single internal calibrant may cause significant systematic error in CCS values, however, we found that the systematic error is only 1.2% and the random error is lower with apo-myoglobin as an internal versus external calibrants (Figure ). The systematic error could be further reduced using multiple internal calibrants instead, but precise CCS values are not necessary for accurate quantitation. Moreover, multiple internal standards could complicate the quantitation. Thus, we decided to use a single internal calibrant.