A New Method for the Isolation of the (-Terminal Peptide of Proteins
185
If all lysine residues are completely blocked, only three tryptic peptides should
be expected. In fact, three major peaks were obtained (Fig. 12.5C). However, it is
also evident that other minor peaks have also been generated by the cleavage of
trypsin in lysine residues that were not completely blocked.
In the selective blocking of proteolytic peptides, the maleic anhydride was used.
The rp-HPLC of the non-retained fraction showed a major peak (Fig. 12.5D). When
it was analyzed by FAB mass spectrometry (Fig. 12.5E), a signal appeared at 1805.9
Da. It matches very well with the theoretical mass value expected (1805.8) for the Cterminal peptide of Cytochrome C, taking into account the presence of two succinyl
lysine residues and a maleyl residue at the N-terminus end. This result indicates that
incomplete blocking of the basic residues in the whole protein is not a limitation to
successful isolation of the C-terminal peptide of the analyzed proteins.
The previous maleyl-blocked peptide was treated with a mixture of acetic and
formic acids and analyzed by automatic Edman degradation (Fig. 12.6). The first
ten amino acids from the N-terminus were clearly determined, including the
succinyl-Iysine residues at the cycles 8 and 9 that eluted between His and Ala in the
PTH amino acid standard. This result confirms that a free amino terminal group
was generated after the acid treatment.
The FAB mass spectrum of the acid-treated peptide showed a signal at 1707.9
Da that matched very well with the theoretical mass value expected (1707.8 Da),
taking into account the presence of two succinyl-Iysine residues and a free amino
terminal group (data not shown). Since no signal of the blocked peptide was
observed in the mass spectra, it indicates that a quantitative de-blocking of the
amino terminal group was obtained. Therefore, our methodology is also compatible with the Edman sequencing particularly when maleyl anhydride is used in the
selective and reversible blocking of the proteolytic peptides.
This methodology was successfully applied to three other proteins myoglobin,
mutated IL-2 and bovine mucorpepsin (Fig. 12.7). In the chromatogram of the
retained fraction several peaks were obtained however in the chromatogram of the
non-retained fraction a major peak was obtained and it always contained the Cterminal peptide. Table 12.1 summarized the FAB-MS analysis of the major fraction
obtained in the rp-HPLC analysis of the non-retained fraction. Amino acid analysis
of the C-terminal peptides was performed and good agreement was found between
the theoretical and experimental amino acid composition (data not shown).
4
Conclusions
This strategy is very easy to implement in any protein chemistry lab. The derivatization reaction, the desalting of the blocked pep tides and cation exchange chromatography for the isolation of the C-terminal peptide can be easily performed.
All the reagents and materials required for the isolation of the C-terminal peptide
are not expensive and are commercially available.
This strategy is compatible with the automatic Edman sequencing and all the
proteolytic peptides can also be recovered for their further analysis or sequencing.
Once the tryptic peptides are selectively derivatized, it is only a matter of separating charged from the neutral species. Using two different chromatographic
185
If all lysine residues are completely blocked, only three tryptic peptides should
be expected. In fact, three major peaks were obtained (Fig. 12.5C). However, it is
also evident that other minor peaks have also been generated by the cleavage of
trypsin in lysine residues that were not completely blocked.
In the selective blocking of proteolytic peptides, the maleic anhydride was used.
The rp-HPLC of the non-retained fraction showed a major peak (Fig. 12.5D). When
it was analyzed by FAB mass spectrometry (Fig. 12.5E), a signal appeared at 1805.9
Da. It matches very well with the theoretical mass value expected (1805.8) for the Cterminal peptide of Cytochrome C, taking into account the presence of two succinyl
lysine residues and a maleyl residue at the N-terminus end. This result indicates that
incomplete blocking of the basic residues in the whole protein is not a limitation to
successful isolation of the C-terminal peptide of the analyzed proteins.
The previous maleyl-blocked peptide was treated with a mixture of acetic and
formic acids and analyzed by automatic Edman degradation (Fig. 12.6). The first
ten amino acids from the N-terminus were clearly determined, including the
succinyl-Iysine residues at the cycles 8 and 9 that eluted between His and Ala in the
PTH amino acid standard. This result confirms that a free amino terminal group
was generated after the acid treatment.
The FAB mass spectrum of the acid-treated peptide showed a signal at 1707.9
Da that matched very well with the theoretical mass value expected (1707.8 Da),
taking into account the presence of two succinyl-Iysine residues and a free amino
terminal group (data not shown). Since no signal of the blocked peptide was
observed in the mass spectra, it indicates that a quantitative de-blocking of the
amino terminal group was obtained. Therefore, our methodology is also compatible with the Edman sequencing particularly when maleyl anhydride is used in the
selective and reversible blocking of the proteolytic peptides.
This methodology was successfully applied to three other proteins myoglobin,
mutated IL-2 and bovine mucorpepsin (Fig. 12.7). In the chromatogram of the
retained fraction several peaks were obtained however in the chromatogram of the
non-retained fraction a major peak was obtained and it always contained the Cterminal peptide. Table 12.1 summarized the FAB-MS analysis of the major fraction
obtained in the rp-HPLC analysis of the non-retained fraction. Amino acid analysis
of the C-terminal peptides was performed and good agreement was found between
the theoretical and experimental amino acid composition (data not shown).
4
Conclusions
This strategy is very easy to implement in any protein chemistry lab. The derivatization reaction, the desalting of the blocked pep tides and cation exchange chromatography for the isolation of the C-terminal peptide can be easily performed.
All the reagents and materials required for the isolation of the C-terminal peptide
are not expensive and are commercially available.
This strategy is compatible with the automatic Edman sequencing and all the
proteolytic peptides can also be recovered for their further analysis or sequencing.
Once the tryptic peptides are selectively derivatized, it is only a matter of separating charged from the neutral species. Using two different chromatographic