Sequence verification of human creatine kinase (43 kDa) isozymes by high-resolution tandem mass spectrometry.
Amino acid sequencing by recombinant DNA technology, although dramatically useful, is subject to base reading errors, is indirect, and is insensitive to posttranslational processing. Mass spectrometry techniques can provide molecular weight data from even relatively large proteins for such cDNA sequences and can serve as a check of an enzyme's purity and sequence integrity. Multiply-charged ions from electrospray ionization can be dissociated to yield structural information by tandem mass spectrometry, providing a second method for gaining additional confidence in primary sequence confirmation. Here, accurate (+/- 1 Da) molecular weight and molecular ion dissociation information for human muscle and brain creatine kinases has been obtained by electrospray ionization coupled with Fourier-transform mass spectrometry to help distinguish which of several published amino acid sequences for both enzymes are correct. The results herein are consistent with one published sequence for each isozyme, and the heterogeneity indicated by isoelectric focusing due to 1-Da deamidation changes. This approach appears generally useful for detailed sequence verification of recombinant proteins.
Wood
T D
TD
Department of Chemistry, Baker Laboratory, Cornell University, Ithaca, NY 14853-1301, USA.
Chen
L H
LH
White
C B
CB
Babbitt
P C
PC
Kenyon
G L
GL
McLafferty
F W
FW
D016477:Artifacts; D001921:Brain / Q000201:enzymology*; D003402:Creatine Kinase / Q000737:chemistry*; D005583:Fourier Analysis; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*
D007527:Isoenzymes; D003402:Creatine Kinase