Rapid identification of calbindin-D28k cyanogen bromide peptide fragments by plasma desorption mass spectrometry.
Chicken intestinal calbindin-D28k is an intracellular protein which is believed to have a fundamental role in vitamin D-mediated transport of calcium. A mapping approach based on 252Cf plasma desorption mass spectrometry (PD mapping) was used to screen the DNA-deduced sequence of calbindin-D28k for sequence changes and post-translational modifications. In the PD mapping experiment, purified calbindin-D28k was cleaved with cyanogen bromide and the resulting peptides were subjected to PD mass spectrometric analysis either as a mixture or as high-performance liquid chromatography isolated fractions. The DNA-derived primary structure of calbindin-D28k was confirmed by rapid PD mass spectral identification of the CNBr peptide fragments, and the nature of the N-terminal blocking group was readily determined to be an acetyl group. The relatively non-destructive nature of the PD mass spectrometric analysis allowed the mapping of the N-terminal peptide through an additional in situ V8 protease enzymatic reaction.
Department of Pharmacology, Mayo Clinic, Rochester, Minnesota 55905.
A
A
Tsarbopoulos
M
M
Gross
R
R
Kumar
I
I
Jardine
D000595:Amino Acid Sequence; D000818:Animals; D064026:Calbindins; D002645:Chickens; D002851:Chromatography, High Pressure Liquid; D003488:Cyanogen Bromide; D004220:Disulfides / Q000032:analysis; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000032:analysis; D064030:S100 Calcium Binding Protein G / Q000032:analysis*; D014357:Trypsin
D064026:Calbindins; D004220:Disulfides; D010446:Peptide Fragments; D064030:S100 Calcium Binding Protein G; D014357:Trypsin; D003488:Cyanogen Bromide