Identification of methionine-containing tryptic peptides of unstable beta-tubulin separated by reverse-phase high-performance liquid chromatography.
Methods for examining altered regions in unstable mutant proteins are described. The strategy is illustrated using assembly defective Chinese hamster beta-tubulin subunits that are rapidly degraded in the cell. These unstable proteins are metabolically labeled to high specific activity and isolated as spots on two-dimensional gels. Conditions for the generation of tryptic peptides from gel pieces containing beta-tubulin and their subsequent resolution by HPLC have been worked out. Through a combination of dual labeling with various tritiated amino acids and [35S]methionine as well as partial sequence analysis, the identification of several HPLC peaks with the known sequence of beta-tubulin has been accomplished. This technique should greatly aid attempts to map the sites of mutational alterations in beta-tubulin polypeptides, and the general strategy should be readily applicable to other mutant proteins.
Minotti
A M
AM
Department of Pharmacology, University of Texas Medical School, Houston 77225.
Loeb
L M
LM
Cook
R
R
Boggs
B A
BA
Cabral
F
F
D000595:Amino Acid Sequence; D000818:Animals; D002460:Cell Line; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006224:Cricetinae; D005260:Female; D008715:Methionine / Q000032:analysis*; D009154:Mutation; D010053:Ovary / Q000166:cytology; D010446:Peptide Fragments / Q000302:isolation & purification*; D010449:Peptide Mapping; D014357:Trypsin / Q000378:metabolism; D014404:Tubulin / Q000032:analysis* / Q000302:isolation & purification
D010446:Peptide Fragments; D014404:Tubulin; D008715:Methionine; D014357:Trypsin