176
L. J. GONZALEZ et aI.
The first method reported for this purpose was the diagonal electrophoresis
(Naughton and Hagopian 1962). This involves the separation of the tryptic peptides on a paper electrophoresis, their treatment with carboxypeptidase B (CPB)
and their further separation in a second dimension. The C-terminal peptide is
not affected by the CPB treatment unless the protein has a basic amino acid at the
C-terminal (Lys/Arg). Therefore, the C-terminal peptide should be the only species that does not change its mobility in either dimension of the electrophoresis,
and will be detected just in the diagonal. It should be noticed that the success of
this methodology depends on the high purity of the CPB preparation, because
contamination with other CPases could also degrade the C-terminal peptide and
affect its mobility in the second dimension.
Another approach is based on the comparative rp-HPLC peptide mapping
between the Cpase-treated and the native protein (Isobe K et a1.l986). The Cterminal peptide should be the only fraction to shift its retention time when both
chromatograms are compared. However, this method has limitations similar to
the strategies that use Cpases and on the other hand, it is difficult to estimate the
extent of the C-terminal degradation when the protein has a C-terminal ragged
end.
Other authors (Rose et al. 1988) have used the stable ISO-labeling of proteolytic peptides to identify the C-terminal peptide of the protein. All internal peptides incorporate ISO at their C-termini during the proteolytic digestion, while
the C-terminal peptide doesn't: This difference can be observed in the shift of the
signals when the mass spectra of the fractions obtained in presence and in
absence of H2 1S 0 are compared. One limitation of this methodology is that the Cterminal peptide of the protein can also be labeled if the protease have affinity for
binding to the C-terminal amino acid of the protein, and this is much more probable with longer digestion times.
This strategy has additional advantages for the sequencing of all internal peptides eSO-labeled species}, since the N- and the C-terminal ions can be easily differentiated in the daughter ion spectra (Takao et al. 1991, Takao et al. 1993). However the above mentioned advantages cannot be utilized to obtain an easier and
more reliable sequencing by mass spectrometry of the C-terminal peptide
because it is a non-labeled species.
Kumazaki et.al in 1986 proposed the affinity chromatography for the isolation
of the C-terminal peptide. The immobilized anhydrotrypsin binds all the tryptic
peptides (end in Lys or Arg) except the C-terminal peptide. In this strategy, the
internal peptides originated by non-specific cleavages of trypsin will also be collected in the same fraction where the C-terminal peptide appears and if the protein ends in lysine or arginine, the C-terminal peptide would also be retained on
the column.
Another strategy has been also developed for the isolation of the C-terminal
peptide after the cyanogen bromide treatment of the protein (Murphy and Fenselau, 1995). It comprises the determination of the increment in mass of all cyanogen bromide peptides after methylation (CH30H/HCI). The C-terminal peptide
should be the only peptide that increases its molecular weight by a multiple of 14
Da because it has a free carboxyl terminal group, while other peptides are transformed into homoserine lactone. This strategy has the disadvantage that it is
L. J. GONZALEZ et aI.
The first method reported for this purpose was the diagonal electrophoresis
(Naughton and Hagopian 1962). This involves the separation of the tryptic peptides on a paper electrophoresis, their treatment with carboxypeptidase B (CPB)
and their further separation in a second dimension. The C-terminal peptide is
not affected by the CPB treatment unless the protein has a basic amino acid at the
C-terminal (Lys/Arg). Therefore, the C-terminal peptide should be the only species that does not change its mobility in either dimension of the electrophoresis,
and will be detected just in the diagonal. It should be noticed that the success of
this methodology depends on the high purity of the CPB preparation, because
contamination with other CPases could also degrade the C-terminal peptide and
affect its mobility in the second dimension.
Another approach is based on the comparative rp-HPLC peptide mapping
between the Cpase-treated and the native protein (Isobe K et a1.l986). The Cterminal peptide should be the only fraction to shift its retention time when both
chromatograms are compared. However, this method has limitations similar to
the strategies that use Cpases and on the other hand, it is difficult to estimate the
extent of the C-terminal degradation when the protein has a C-terminal ragged
end.
Other authors (Rose et al. 1988) have used the stable ISO-labeling of proteolytic peptides to identify the C-terminal peptide of the protein. All internal peptides incorporate ISO at their C-termini during the proteolytic digestion, while
the C-terminal peptide doesn't: This difference can be observed in the shift of the
signals when the mass spectra of the fractions obtained in presence and in
absence of H2 1S 0 are compared. One limitation of this methodology is that the Cterminal peptide of the protein can also be labeled if the protease have affinity for
binding to the C-terminal amino acid of the protein, and this is much more probable with longer digestion times.
This strategy has additional advantages for the sequencing of all internal peptides eSO-labeled species}, since the N- and the C-terminal ions can be easily differentiated in the daughter ion spectra (Takao et al. 1991, Takao et al. 1993). However the above mentioned advantages cannot be utilized to obtain an easier and
more reliable sequencing by mass spectrometry of the C-terminal peptide
because it is a non-labeled species.
Kumazaki et.al in 1986 proposed the affinity chromatography for the isolation
of the C-terminal peptide. The immobilized anhydrotrypsin binds all the tryptic
peptides (end in Lys or Arg) except the C-terminal peptide. In this strategy, the
internal peptides originated by non-specific cleavages of trypsin will also be collected in the same fraction where the C-terminal peptide appears and if the protein ends in lysine or arginine, the C-terminal peptide would also be retained on
the column.
Another strategy has been also developed for the isolation of the C-terminal
peptide after the cyanogen bromide treatment of the protein (Murphy and Fenselau, 1995). It comprises the determination of the increment in mass of all cyanogen bromide peptides after methylation (CH30H/HCI). The C-terminal peptide
should be the only peptide that increases its molecular weight by a multiple of 14
Da because it has a free carboxyl terminal group, while other peptides are transformed into homoserine lactone. This strategy has the disadvantage that it is