A New Method for the Isolation of the (-Terminal Peptide of Proteins
177
cyanogen bromide dependent, and it requires special handling. On the other
hand, pep tides originated by the non-specific cleavages of the cyanogen bromide,
or others that could be originated by the drastic acidic conditions used in this
chemical cleavage, will exhibit the same behavior as the C-terminal peptide during the methylation analysis.
The cation exchange chromatography could be used to isolate the C-terminal
peptide in the complex mixture generated by a proteolytic digestion. However, it
is not easy to find unique and reproducible conditions that would allow the isolation of the C-terminal peptide in all proteins. In 1993, Gorman and Shiell published the use of cation exchange chromatography to successfully isolate, simultaneously, the C- and the N-terminal peptides of N-terminal blocked proteins.
In this paper, we report on a new strategy for the selective isolation of the Cterminal peptide of proteins by using the combination of the highly specific
chemical derivatization of the N-terminal end of the tryptic peptides (Wetzel et
al. 1990, Stults et al. 1993) and cation exchange chromatography. By this procedure, the tryptic peptides are transformed at acid pH, into a mixture of singlecharge and neutral peptides, which can be easily separated by cation exchange
chromatography.
2
Procedures
2.1
Selective Blocking of NH2-Terminal Groups
The peptide mixture was dissolved in buffer MES (300 mM, pH = 6.0) containing
Glycine 60 mM to reach a final concentration of 130-150 [lM.
Fifty-fold molar excess of the organic acid anhydride (dissolved in dry THF)
with respect to the amount of amino terminal groups generated during the proteolytic digestion, was added. The reaction time for acetic anhydride was only
3 minutes and for the other anhydrides, 10 to 15 minutes were required. The
reaction was maintained at O°C using an ice bath with an efficient stirring inside
the reaction vial.
2.2
Desalting of N-Terminal Blocked Peptides
The peptide mixture was desalted using a mini-column (2 x 0.2 cm) packed with
C4 widepore material (VYDAC). A linear gradient from 0 to 80 % of CH3CN
(0.05 % of TFA) in 10 minutes was used for desalting the derivatized peptides.
The pool of blocked pep tides was detected by monitoring the absorbance at
226nm.
177
cyanogen bromide dependent, and it requires special handling. On the other
hand, pep tides originated by the non-specific cleavages of the cyanogen bromide,
or others that could be originated by the drastic acidic conditions used in this
chemical cleavage, will exhibit the same behavior as the C-terminal peptide during the methylation analysis.
The cation exchange chromatography could be used to isolate the C-terminal
peptide in the complex mixture generated by a proteolytic digestion. However, it
is not easy to find unique and reproducible conditions that would allow the isolation of the C-terminal peptide in all proteins. In 1993, Gorman and Shiell published the use of cation exchange chromatography to successfully isolate, simultaneously, the C- and the N-terminal peptides of N-terminal blocked proteins.
In this paper, we report on a new strategy for the selective isolation of the Cterminal peptide of proteins by using the combination of the highly specific
chemical derivatization of the N-terminal end of the tryptic peptides (Wetzel et
al. 1990, Stults et al. 1993) and cation exchange chromatography. By this procedure, the tryptic peptides are transformed at acid pH, into a mixture of singlecharge and neutral peptides, which can be easily separated by cation exchange
chromatography.
2
Procedures
2.1
Selective Blocking of NH2-Terminal Groups
The peptide mixture was dissolved in buffer MES (300 mM, pH = 6.0) containing
Glycine 60 mM to reach a final concentration of 130-150 [lM.
Fifty-fold molar excess of the organic acid anhydride (dissolved in dry THF)
with respect to the amount of amino terminal groups generated during the proteolytic digestion, was added. The reaction time for acetic anhydride was only
3 minutes and for the other anhydrides, 10 to 15 minutes were required. The
reaction was maintained at O°C using an ice bath with an efficient stirring inside
the reaction vial.
2.2
Desalting of N-Terminal Blocked Peptides
The peptide mixture was desalted using a mini-column (2 x 0.2 cm) packed with
C4 widepore material (VYDAC). A linear gradient from 0 to 80 % of CH3CN
(0.05 % of TFA) in 10 minutes was used for desalting the derivatized peptides.
The pool of blocked pep tides was detected by monitoring the absorbance at
226nm.