High performance capillary electrophoresis - T.E.A.M.

Modes
a) CH 3COONa-CH 3COOH
difficult to analyze by the traditional techniques of slab gel
electrophoresis, isoelectric focusing, or liquid chromatography.
In some cases CZE is advantageous. Shown in figure 27
is the separation of glycoforms of the human recombinant
protein hormone, erythropoietin. The different species result
from heterogeneity after post-translational modification. CZE
is well suited for such analyses since many post translational
modifications have an impact on protein charge (that is, N-
or C- terminal modifications, phosphorylation, carboxylation,
or N-glycosylation).
b) CH 3COONa-H 3PO
c) CH 3COONa-H 2SO4
0 5 10 15
Time [min]
Figure 27
Influence of buffer composition on
separation of erythropoietin glycoforms 13
Conditions: Buffer concentration = 100 mM,
pH 4, V = 10 kV, i = 10, 120, 200 mA
in a, b, and c, respectively,
l = 20 cm, L = 27 cm, id = 75 mm,
l = 214 nm
Significant success has been realized for peptide mapping
by CZE. In peptide mapping a protein is enzymatically or
chemically cleaved into smaller peptide fragments and
subsequently separated. The analysis is primarily qualitative
and is used to detect subtle differences in proteins. A typical
CZE peptide map is shown in figure 28. CZE is also useful as
a second-dimension analysis of HPLC-purified peptides
(figure 29).
mAU
30
28
n
20
Reproducibility (RSD %)
Migration time
2.5%
Mobility
0.3%
26
24
Figure 28
Rapid BSA peptide map
Conditions: 20 mM phosphate, pH 7, V = 25 kV,
i = 16 mA, l = 50 cm, L = 57 cm,
id = 50 mm with 3X extended
pathlength detection cell,
l = 200 nm
22
20
18
5 6 7 8 9 10 11 12
Time [min]
59