Enzymatic and Chemical Deblocking of N-Terminally Modified Proteins
313
from the membrane. For better elution of pep tides and to reduce hydrophobic
interaction between peptide and PVDF membrane, 10 % acetonitrile is added in
the digestion buffer. Non blocked pep tides are reacted with PITC in the next step
and form phenylthiocarbamyl pep tides, which are oxidized to phenylcarbamyl
peptides and are not detected during subsequent Edman degradation. The acetylated amino acid of the N-terminal peptide is selectively removed by digestion
with AARE.
The above described method works very well for pep tides smaller than 10 residues. The extraction from the PVDF membrane for larger peptides is difficult. To
overcome this problem, second digestion with another protease may be performed using the same membrane.
Instead of reaction with PITC, the non blocked pep tides after protease digestion can be succinylated and the N-terminal blocked peptide is then deblocked
with AARE (Krishna et al. 1991).
After releasing of N-terminaly blocked amino acid by AARE, the blocked residue can be identified by mass spectrometry.
The efficiency of deblocking and sequencing depends primarily on tryptic
digestion and elution yields from the PVDF membrane.
2.4
Deblocking of Myristoyl Group
Mirystoyl group of the blocked proteins can be removed when peptide N-fatty
acylase (WAKO Pure Chemicals) is used. The procedure can be use as described
for AARE.
Also treatment with deblocking aminopeptidase (DAP) can be applied for deblocking of mirystoyl groups. Optimized conditions see under DAP (see chapter 2.1).
3
Chemical Deblocking Methods
3.1
Deblocking of Proteins with N-terminal Acetylserine and Acetylthreonine
Proteins with acetylserine and acetylthreonine can be deblocked by trifluorocetic
acid (TFA) treatment (Wellner et al. 1990). Comparison with all N-blocked proteins shows, that 36 % of known acetylated amino acids are acetylserine and 4 %
acetylthreonine. The yields of chemical deblocking reaction with TFA vary
depending on proteins by up to 40 %. The protein sample is first applied to the
glass fiber fIlter, dried and saturated with anhydrous TFA. The deblocking reaction is performed for 2-3 days. The possible mechanism proposed by Wellner
1990 of the deblocking reaction is shown in Fig. 22.6.
The advantage of this method is that deblocking is simple and rapid, only the
sequencing initial yields of proteins treated with TFA are rather low, about 7 %
(Wellner et al. 1990).
The deblocking reaction can be performed in solution or on the membrane
(Hirano 1997). When the membrane e.g PVDF membrane is exposed to TFA
313
from the membrane. For better elution of pep tides and to reduce hydrophobic
interaction between peptide and PVDF membrane, 10 % acetonitrile is added in
the digestion buffer. Non blocked pep tides are reacted with PITC in the next step
and form phenylthiocarbamyl pep tides, which are oxidized to phenylcarbamyl
peptides and are not detected during subsequent Edman degradation. The acetylated amino acid of the N-terminal peptide is selectively removed by digestion
with AARE.
The above described method works very well for pep tides smaller than 10 residues. The extraction from the PVDF membrane for larger peptides is difficult. To
overcome this problem, second digestion with another protease may be performed using the same membrane.
Instead of reaction with PITC, the non blocked pep tides after protease digestion can be succinylated and the N-terminal blocked peptide is then deblocked
with AARE (Krishna et al. 1991).
After releasing of N-terminaly blocked amino acid by AARE, the blocked residue can be identified by mass spectrometry.
The efficiency of deblocking and sequencing depends primarily on tryptic
digestion and elution yields from the PVDF membrane.
2.4
Deblocking of Myristoyl Group
Mirystoyl group of the blocked proteins can be removed when peptide N-fatty
acylase (WAKO Pure Chemicals) is used. The procedure can be use as described
for AARE.
Also treatment with deblocking aminopeptidase (DAP) can be applied for deblocking of mirystoyl groups. Optimized conditions see under DAP (see chapter 2.1).
3
Chemical Deblocking Methods
3.1
Deblocking of Proteins with N-terminal Acetylserine and Acetylthreonine
Proteins with acetylserine and acetylthreonine can be deblocked by trifluorocetic
acid (TFA) treatment (Wellner et al. 1990). Comparison with all N-blocked proteins shows, that 36 % of known acetylated amino acids are acetylserine and 4 %
acetylthreonine. The yields of chemical deblocking reaction with TFA vary
depending on proteins by up to 40 %. The protein sample is first applied to the
glass fiber fIlter, dried and saturated with anhydrous TFA. The deblocking reaction is performed for 2-3 days. The possible mechanism proposed by Wellner
1990 of the deblocking reaction is shown in Fig. 22.6.
The advantage of this method is that deblocking is simple and rapid, only the
sequencing initial yields of proteins treated with TFA are rather low, about 7 %
(Wellner et al. 1990).
The deblocking reaction can be performed in solution or on the membrane
(Hirano 1997). When the membrane e.g PVDF membrane is exposed to TFA