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Alcoholytic deblocking of N‐terminally acetylated peptides and proteins for sequence analysis
Author(s) -
Bergman Tomas,
Gheorghe Madalina T.,
Hjelmqvist Lars,
Jörnvall Hans
Publication year - 1996
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(96)00657-6
Subject(s) - edman degradation , deblocking filter , acetylation , trifluoroacetic acid , cleavage (geology) , chemistry , methanol , peptide , peptide sequence , sequence (biology) , chromatography , combinatorial chemistry , biochemistry , organic chemistry , biology , computer science , paleontology , fracture (geology) , computer vision , gene
N‐terminal acetylation of polypeptides is a common feature preventing direct Edman degradation. We describe a method for the removal of the acetyl group, with only a low extent of internal peptide bond cleavage, also in large proteins, by treatment at room temperature with trifluoroacetic acid and methanol. The alcohol is essential for selective deacetylation, and it is proposed that the deblocking mechanism consists of an acid‐catalyzed nucleophilic substitution involving methanol. The extent of deacetylation is limited, but the initial yield in the sequence analysis can be up to 10%. Deblocking of samples spotted or blotted onto sequencer filters is equally possible as the use of isolated samples from column separations. Deblocking on sequencer filters is also possible directly after negative results on initial sequencer attempts with samples proving to be blocked.