Direct Sequence Data from Heterogeneous Creatine Kinase (43 kDa) by High-Resolution Tandem Mass Spectrometry

Isoelectric focusing separation of recombinant rabbit muscle creatine kinase (CK) and its ²⁸²Cys → 282Ser mutant shows the presence of three and two isoforms, respectively, that exhibit equivalent enzymatic activity. Electrospray ionization coupled with Fourier-transform mass spectrometry (105 resolving power) of both CKs indicates that their major components are within ±2 Da of the Mr value predicted from the cDNA sequences of these mixtures. Dissociation of (M + nH)ⁿ⁺ gives no evidence that the components of either CK are isomers; the masses of the 51 fragment ions correlate completely (±1 Da) with the values predicted from the cDNA sequence and confirm the identities of 21 of the 380 amino acids and the ²⁸²Cys -- ²⁸²Ser replacement in the mutant. The results are consistent with one or two steps of post-translational amidation/deamidation (NH₂ → OH, 16 Da → 17 Da), each of which would produce only a 1 Da difference in Mr, with the fragment masses indicating that at least one modification occurs between residues 212 and 282.