Enzymatic and Chemical Deblocking of N-Terminally Modified Proteins
317
ble, deblocking with AARE is performed to remove acetylamino acids_ If
sequencing still fails, other methods should be used or developed. Fig. 22.7 shows
the strategy of sequencial deblocking for N-terminally blocked proteins electroblotted onto a PVDF-membrane.
5
Sample Preparation of Minute Amounts of Proteins
If the N-terminally blocked protein is available only in picomole amounts, the
biotin-avidin method may be used to remove selectively the blocked N-terminal
peptide (Mitsunaga et al. 1993).
After digestion of N-terminally blocked proteins with lysylendopeptidase, the
resulted peptides were digested in the second step with carboxypeptidase B to
remove the C-terminallysine from each peptide. In the next reaction, the peptides were bound to phenylendiisothiocyanate (DITC) glass to eliminate the peptides and lysines except for the N-terminal blocked peptides.
The unabsorbed peptides by DITC glass treatment were reacted with the Nhydrosuccinimide-biotin to biotinylate the a- and E-amino groups of remained
peptides and lysines. The biotinylated pep tides and lysine residues were immobilized on an avidin-agarose column and the N-terminally blocked peptides were
obtained as an unabsorbed fraction, which can be futher applied to Edman degradation or mass spectrometry. Fig. 22.8 shows the scheme for above described
isolation of the N-terminal peptide from N-terminally blocked proteins.
6
Conclusion
Summarizing the methods for deblocking of N-terminally modified proteins,
several enzymes or chemicals can be used for the cleavage of different blocking
groups from N-terminal end. The above mentioned methods can be used for
deblocking in solution or on the inert membrane, which may be used directly for
subsequent amino acid sequence analysis. Although yields may still vary,
depending on the sequence, blocking groups, and molecular weight, the above
methods can be applied as a convenient way of deblocking posttranslationally
modified N-terminal amino acids. It is important to choose a suitable method for
the deblocking dependent of the blocking group, because no generally applicable
method for deblocking has yet been developed.
7
Acknowledgment
We would like to thank Dr. Susumi Tsunasawa Takara Shuzo, Japan for enthusiastic meetings, valuable discussion and for sequencing of SOD.
317
ble, deblocking with AARE is performed to remove acetylamino acids_ If
sequencing still fails, other methods should be used or developed. Fig. 22.7 shows
the strategy of sequencial deblocking for N-terminally blocked proteins electroblotted onto a PVDF-membrane.
5
Sample Preparation of Minute Amounts of Proteins
If the N-terminally blocked protein is available only in picomole amounts, the
biotin-avidin method may be used to remove selectively the blocked N-terminal
peptide (Mitsunaga et al. 1993).
After digestion of N-terminally blocked proteins with lysylendopeptidase, the
resulted peptides were digested in the second step with carboxypeptidase B to
remove the C-terminallysine from each peptide. In the next reaction, the peptides were bound to phenylendiisothiocyanate (DITC) glass to eliminate the peptides and lysines except for the N-terminal blocked peptides.
The unabsorbed peptides by DITC glass treatment were reacted with the Nhydrosuccinimide-biotin to biotinylate the a- and E-amino groups of remained
peptides and lysines. The biotinylated pep tides and lysine residues were immobilized on an avidin-agarose column and the N-terminally blocked peptides were
obtained as an unabsorbed fraction, which can be futher applied to Edman degradation or mass spectrometry. Fig. 22.8 shows the scheme for above described
isolation of the N-terminal peptide from N-terminally blocked proteins.
6
Conclusion
Summarizing the methods for deblocking of N-terminally modified proteins,
several enzymes or chemicals can be used for the cleavage of different blocking
groups from N-terminal end. The above mentioned methods can be used for
deblocking in solution or on the inert membrane, which may be used directly for
subsequent amino acid sequence analysis. Although yields may still vary,
depending on the sequence, blocking groups, and molecular weight, the above
methods can be applied as a convenient way of deblocking posttranslationally
modified N-terminal amino acids. It is important to choose a suitable method for
the deblocking dependent of the blocking group, because no generally applicable
method for deblocking has yet been developed.
7
Acknowledgment
We would like to thank Dr. Susumi Tsunasawa Takara Shuzo, Japan for enthusiastic meetings, valuable discussion and for sequencing of SOD.