title: Direct sequence data from heterogeneous creatine kinase (43 kDa) by high-resolution tandem mass spectrometry.
abstract: 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 (10(5) resolving power) of both CKs indicates that their major components are within +/- 2 Da of the M(r) 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 M(r), with the fragment masses indicating that at least one modification occurs between residues 212 and 282.
authors.affiliation: Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, New York 14853-1301, USA.
authors.forename: T D
authors.initials: TD
authors.lastname: Wood
authors.forename: L H
authors.initials: LH
authors.lastname: Chen
authors.forename: N L
authors.initials: NL
authors.lastname: Kelleher
authors.forename: D P
authors.initials: DP
authors.lastname: Little
authors.forename: G L
authors.initials: GL
authors.lastname: Kenyon
authors.forename: F W
authors.initials: FW
authors.lastname: McLafferty
mesh_terms: D000818:Animals; D003402:Creatine Kinase / Q000737:chemistry*; D005583:Fourier Analysis; D007527:Isoenzymes / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008970:Molecular Weight; D009132:Muscles / Q000201:enzymology; D009154:Mutation; D011499:Protein Processing, Post-Translational; D011817:Rabbits; D017421:Sequence Analysis / Q000379:methods*
chemical_list: D007527:Isoenzymes; D003402:Creatine Kinase