Additional possible tools for identification of proteins on one- or two-dimensional electrophoresis.
Additional, essentially chemical, identification methods of proteins in polyacrylamide gel electrophoresis are described. Two cleavages of peptide bonds were used at the C-side of aspartic acid with a 0.2% pentafluoropropionic acid (PFPA) aqueous vapor at 90 degrees C for 4-16 h, and the N-side of serine/threonine with an S-ethyl trifluorothioacetate vapor at 50 degrees C for 6-24 h. The products were analyzed by mass spectrometry-peptide mass fingerprinting. A new type of C-terminal sequencing at multisites of protein was introduced. An aqueous vapor of 90% PFPA at 90 degrees C for 2-16 h provided cleavages at the C-side of aspartic acid and the N-side of serine/threonine and simultaneous successive truncation at the C-termini of the cleaved fragments. The product resulted in C-terminal sequences at multisites in proteins by mass spectrometric analysis. The following chemical deblocking methods were used. Anhydrous hydrazine vapor at -5 degrees C for 8 h deblocked the N-formyl group, and the vapor at 20 degrees C for 4 h deblocked pyrrolidone carboxylate. N-acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50 degrees C for 1 h, followed by reaction with p-sulfophenylisothiocyanate at pH 6.0. These methods were applied to a variety of protein spots on polyacrylamide gels. A new stepwise C-terminal sequencing of protein from polyacrylamide gels is also described.
Tsugita
A
A
Research Institute for Biosciences, Science University of Tokyo, Yamazaki, Noda, Japan. tsugita@jipdalph.rb.noda.sut.ac.jp
Kamo
M
M
Miyazaki
K
K
Takayama
M
M
Kawakami
T
T
Shen
R
R
Nozawa
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001224:Aspartic Acid / Q000032:analysis; D002256:Carbonic Anhydrases / Q000737:chemistry; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000295:instrumentation / Q000379:methods*; D005934:Glucagon / Q000737:chemistry; D006801:Humans; D013058:Mass Spectrometry / Q000295:instrumentation / Q000379:methods; D008969:Molecular Sequence Data; D009113:Muramidase / Q000737:chemistry; D010449:Peptide Mapping / Q000379:methods; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D017421:Sequence Analysis / Q000379:methods; D012694:Serine / Q000032:analysis; D013912:Threonine / Q000032:analysis
D011506:Proteins; D013912:Threonine; D001224:Aspartic Acid; D012694:Serine; D005934:Glucagon; D009113:Muramidase; D002256:Carbonic Anhydrases