Alcoholytic deblocking of N-terminally acetylated peptides and proteins for sequence analysis.
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.
Bergman
T
T
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden.
Gheorghe
M T
MT
Hjelmqvist
L
L
Jörnvall
H
H
D000107:Acetylation; D000595:Amino Acid Sequence; D000818:Animals; D000432:Methanol; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry*; D017421:Sequence Analysis / Q000379:methods*; D014269:Trifluoroacetic Acid
D010455:Peptides; D011506:Proteins; D014269:Trifluoroacetic Acid; D000432:Methanol