C-terminal sequencing method for proteins in polyacrylamide gel by the reaction of acetic anhydride.
A successive C-terminal amino acid truncation reaction with acetic anhydride was applied on proteins in polyacrylamide gel. Protein bands separated by conventional SDS-PAGE were excised, partially fixed in the gel with glutaraldehyde ethanol solution, dehydrated with ACN and subjected to the truncation reaction with acetic anhydride formamide solution. Pre-treatment of the gel with pyridine aqueous solution was found to enhance the truncation reaction yields. After the truncation reaction, the products were treated with an aqueous solution of dimethylaminoethanol to hydrolyze oxazolone rings at the C termini of the truncated products and O-acetylated products of serine, threonine and/or tyrosine. Several commercially available proteins of 10-40 kDa, as determined by SDS-PAGE, such as myoglobin, trypsin inhibitor, alpha-hemolysin, cytochrome c, chymotrypsin C chain, elastase, acylase and histone H4, were subjected to the C-terminal analysis. The truncated proteins were in-gel digested with trypsin and the extracted peptides were analyzed by MALDI-TOF MS, giving rise to a series of molecular mass ions of the C-terminal truncated fragments corresponding to the C-terminal amino acid sequence of the relevant protein.
Miyazaki
Kenji
K
Proteomics Research Center, Fundamental and Environmental Research Laboratories, NEC Corp., Tsukuba, Japan.
Tsugita
Akira
A
D000089:Acetic Anhydrides; D000595:Amino Acid Sequence; D000818:Animals; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005976:Glutaral; D006736:Horses; D008969:Molecular Sequence Data; D009211:Myoglobin / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D011725:Pyridines; D020539:Sequence Analysis, Protein / Q000379:methods; D013025:Glycine max / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014361:Trypsin Inhibitors / Q000737:chemistry
D000089:Acetic Anhydrides; D009211:Myoglobin; D010446:Peptide Fragments; D011506:Proteins; D011725:Pyridines; D014361:Trypsin Inhibitors; C031800:acetic anhydride; C023666:pyridine; D005976:Glutaral