ISSN:
0173-0835
Keywords:
Multiple C-terminal sequencing at multisites
;
Asp-C cleavage
;
Ser/Thr-N cleavage
;
Deblocking
;
C-terminal sequencing
;
Chemistry
;
Biochemistry and Biotechnology
Source:
Wiley InterScience Backfile Collection 1832-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Additional, essentially chemical, identification methods of proteins in polyacry-lamide 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° for 4-16 h, and the N-side of serine/threonine with an S-ethyl trifluorothioacetate vapor at 50° 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° 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° for 8 h deblocked the N-formyl group, and the vapor at 20° for 4 h deblocked pyrrolidone carboxylate. N-acetylserine/threonine was deblocked by aqueous vapor of 75% PFPA at 50° for 1 h, followed by reaction with p-suifophenylisothiocyanate 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.
Additional Material:
4 Ill.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1002/elps.1150190608
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