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.

Creatine kinase: Essential arginine residues at the nucleotide binding site identified by chemical modification and high-resolution tandem mass spectrometry

“…The mass spectrum of native rabbit muscle CK exhibits an isotopic cluster corresponding to a relative molecular weight (M r ) of 42,982, consistent with its DNA-derived sequence and limited deamidation (2). The spectrum of phenylglyoxalderivatized CK (Fig.…”

“…Previous tandem ESI-FTMS of (M ϩ nH) nϩ ions from rabbit muscle CK gave 52 fragment ions whose masses can be assigned to the cDNA-derived sequence (2). Here, NS dissociation of the derivatized CK (M ϩ nH) nϩ ions was used to identify fragment ions increased in mass by 116 Da and thus modified by phenylglyoxal.…”

“…The ESI-generated ions were transported by three radio-frequency-only quadrupoles through five stages of differential pumping to the trapped ion cell (10 Ϫ9 Torr, 5-s accumulation) in a modified 6.1 Tesla Finnigan FT͞MS-2000 (21). MS͞MS experiments were performed by using nozzle͞skimmer (NS) (22) or sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) (23,24) of charge states isolated by stored waveform inverse FT (SWIFT) (25) under conditions previously described (2). Although the resolving power nominally was 10 5 , in the complex product mixture the 43-kDa molecular ion isotopic peaks were not resolved after deconvolution (26,27), so that their centroid masses were used.…”

“…Mechanistic studies of CK have been made more difficult by the microheterogeneity found in enzyme preparations (1); mass spectrometry (MS) recently has shown (2) that deamidation is a major cause of this. Microheterogeneity also has been an obstacle to obtaining x-ray crystal structures for dimeric cytosolic (muscle, brain, or muscle-brain heterodimer) isoforms (1,2), although recently Fritz-Wolf et al (3) have succeeded in obtaining a 3.0 Å x-ray crystal structure for the octameric chicken mitochondrial CK. Several studies have been done to identify the roles of amino acid residues involved in substrate binding and catalysis in CK.…”

From large analogical instruments to small digital black boxes: 40 years of progress in mass spectrometry and its role in proteomics. Part II 1985–2000

“…This was demonstrated for Creatine Kinase, a 43 kDa protein [102] and also for Thiaminase I (42 kDa) and albumin (67 kDa).…”