Fig. 4. Modification of Glu269 by carbodiimides. (A) Reaction of carbodiimide with carboxyl side-chain. Nucleophilic attack of a carbodiimide on a protonated carboxyl side-chain leads to the formation of O-acyl-isourea with a change in molecular weight (Δ molecular weight) followed by rearrangement to form an N-acylurea with no further change in mass. (B) Collision-induced dissociation spectrum of ²⁶⁸-GELLNASIM-²⁷⁶ and ²⁶⁸-GELLNASIM-²⁷⁶ modified with DiPC. Annotated peaks in the spectra include the singly charged parents (m/z 899.7 and 1025.7, respectively) and the b- and y-ion series. (C) Time course of ²⁶⁸-GELLNASIM-²⁷⁶ modification by carbodiimides with different solubilities. Purified LacY (∼20 µM) in DDM at pH 6.0 was incubated with 20 mM of the following carbodiimides at 30°C leading to the mass shifts shown in parentheses: DiPC (Δ126), DCCD (Δ206), CMC (Δ251) and EDC (Δ155). The total ion current from peptide ²⁶⁸-GELLNASIM-²⁷⁶ modified by carbodiimide divided by the total ion current from unmodified and modified ²⁶⁸-GELLNASIM-²⁷⁶ was used to calculate the percentage modification of Glu269. No measurable difference in the ionization efficiency of the unmodified and modified peptides was detected. Each point represents the average of two independent experiments. Maximum peak height in the absence of modification was ∼5 × 10⁵ counts.