Figure 4

Mass spectrometry reveals conjugation of cucurbitacin to thiols. A) Mass of Cofilin1 protein alone or following incubation with cucurbitacin D, E or I as indicated, as determined by mass spectrometry. B) Mass/charge determination of cucurbitacin E that had been incubated with DTT reveals that a reaction product that conserves mass had been formed, consistent with a Michaels addition reaction. C) MS-MS spectra of the tryptic peptide containing Cys39, modified (left) and unmodified (right) with cucurbitacin E. Correspondent mass of the precursors peptides are indicated in the figures, and highlight that the same peptide sequence has a difference in molecular weight, corresponding to one cucurbitacin E molecule (556.3 Da).