Fig. 2. LC-MS analyses of the wild-type and C191S ICL1 constructs. (a) Mass spectrum of the wild-type ICL1. The calculated molecular mass of the intact protein is 49 639.3 Da (observed: 49 642.1 Da). (b) Mass spectrum of the wild-type ICL1 pre-incubated with itaconate and Mg²⁺. The calculated molecular mass of the wild-type ICL-itaconate covalent adduct is 49 769.4 Da (observed: 49 772.4 Da). (c) Mass spectrum of C191S ICL1 construct with a calculated molecular mass 49 233.9 Da (observed: 49 104.2 Da). The difference between the observed and calculated molecular mass is likely due to cleavage of the N-terminal methionine by the Escherichia coli methionine aminopeptidase.³⁸ (d) Mass spectrum of C191S ICL1 pre-incubated with itaconate and Mg²⁺. No changes were observed between the samples with or without pre-incubation with itaconate and Mg²⁺.