Figure 2.

Noncoded amino acid substitution in CaM. Electrospray ionization mass spectra of (A) wt-CaM, (B) Nle-CaM, and (C) Eth-CaM show the level and distribution of Nle or Eth incorporation in place of the nine Met residues of the wild-type CaM protein. Nle is 18 Da smaller than Met, and the Nle-CaM spectrum (panel B) shows a series of protein masses that decrease as the number of incorporated Nle residues increases. Peaks can be clearly seen for species containing from three to nine Nle substitutions, with the most abundant species at 16,542 Da representing CaM with all nine of its Met residues replaced with Nle. The extra peak at 16,525 represents a loss of NH₃ from the +9 Nle species. On the other hand, Eth is 14 Da larger than Met, and the Eth-CaM spectrum (panel C) shows a series of protein masses that increase with increasing incorporation levels of Eth. The +7 through +9 species are the most abundant, with the fully Eth-substituted protein seen at 16,832 Da. Samples were analyzed using a VG Quattro (Micromass, Manchester, UK) ESI triple quadrupole mass spectrometer.