Optimized alcoholytic deacetylation of N-acetyl-blocked polypeptides for subsequent Edman degradation.
N-terminal protein acetylation is a common posttranslational modification, blocking Edman degradation during sequencer analysis. Use of mass spectrometry allows the analysis also of acetyl-blocked polypeptides; however, for large proteins mass spectrometry is not always informative, and deacetylation by chemical pretreatments is desirable for making direct sequencer analysis possible. For this purpose, alcoholytic deacetylation is attractive. In the present work, we have studied the optimal conditions for specific removal of the acetyl group without extensive cleavage of peptide bonds in general. We find that incubation with trifluoroacetic acid in methanol (1:1, by volume) at an elevated temperature ( approximately 47 degrees C) for 2-3 days results in efficient deacetylation allowing direct application to sequencer analysis with initial yields up to approximately 50% of the amount applied for deblocking. Deacetylation compared to internal peptide bond cleavage is often high, as evaluated by recoveries of residues from the deblocked sequence over those from the background, and this applies to both peptides (up to the order of 10:1 for the specific residue versus the background) and proteins (>2:1). Although yields may still vary and some sequences be only partly susceptible to the chemistry, this deblocking can in many cases allow unambiguous interpretation of N-terminally acetyl-blocked sequences.
Gheorghe
M T
MT
Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, S-171 77, Sweden.
Jörnvall
H
H
Bergman
T
T
D000107:Acetylation; D000426:Alcohol Dehydrogenase / Q000737:chemistry; D000595:Amino Acid Sequence; D019075:Electrophoresis, Capillary; D007700:Kinetics; D013058:Mass Spectrometry; D000432:Methanol; D009943:Organophosphorus Compounds; D010446:Peptide Fragments / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D011506:Proteins / Q000737:chemistry; D017421:Sequence Analysis / Q000379:methods; D013696:Temperature; D014269:Trifluoroacetic Acid
C043517:2-(4-isothiocyanatophenoxy)-1,3,2-dioxaphosphinene 2-oxide; D009943:Organophosphorus Compounds; D010446:Peptide Fragments; D010455:Peptides; D011506:Proteins; D014269:Trifluoroacetic Acid; D000426:Alcohol Dehydrogenase; D000432:Methanol