APPLICATION
NOTE
AN032505-28
StyrosZymeä
Pepsin, Immobilized Enzyme on Polymeric Hard Gel Stationary Phase: On line
digestion of Cytochrome c From Horse Heart and Bovine Heart.
The chemical and mechanical stability of
polymeric hard gel media that are the features of StyrosZymeä
immobilized enzyme columns, allow their use on line in a flow through setting.
The advantages resulting from such
setting are numerous:
·
Digestion time is reduced to a few minutes as
compared to hours.
·
The enzyme cartridge can be used as a direct inlet
to either a LC or a MS system for the analysis of the resulting peptides,
substantially reducing and simplifying the sample handling process and allowing
it to be fully automated.
·
The extent of digestion can be controlled by
changing the flow rate and the temperature. It can also be made fully
reproducible.
·
The immobilized enzyme displays high stability
towards low pH’s, high flow rates, temperatures and back pressures.
·
The possibility of using fast flow rates allows
the cartridge to be reconditioned quickly, further reducing the process time.
·
No autodigestion occurs due to the absence of
contact between enzyme molecules in the immobilized format.
·
A single cartridge can be used during many
digestions without loosing its activity.
·
The ratio of Enzyme to Substrate can be controlled
in order to obtain target peptide in different concentrations.
In the following examples Cytochrome c
from two different sources were used to highlight the variations in peptides
profiles obtained during reversed phase mapping of the resulting digests.
Similar conditions are used in both
cases to achieve over 99% digestion.
A typical digestion consists of running
a known amount of protein in the StyrosZymeä
Pepsin column at flow rates of 50 µl/min (18 cm/hr for a 4.6 mm ID column). The
resulting peptide digests are dumped into a reversed phase column connected in
series with the StyrosZymeä Pepsin
column.
The enzyme column is then taken off line
and the peptide mapped following a gradient.
Chromatogram 1
Peptide digests from Cytochrome c from horse heart separated on a
STYROSä
2R/XH 4.6 X 150 mm at 0.5 ml/min (180 cm/hr)
Chromatogram 2
Peptide digests from Cytochrome c from bovine heart separated on a
STYROSä
2R/XH 4.6 X 150 mm at 0.5 ml/min (180 cm/hr)
Table 1. Operating parameters for the
chromatograms.
| HPLC System |
Agilent 1100, Standard Cell |
| Columns |
StyrosZymeä
Pepsin 4.6 x 33 mm
STYROSä
2R/XH 4.6 X 150 mm |
| Mobile Phase For reversed phase. |
A: 0.075% TFA in H2O
B: 0.075% TFA in ACN:H2O (95:5) |
| Mobile Phase For Digestion. |
100 mM AcONa + 150 mM NaCl in DI H2O at
pH=4.5. |
| Detection: |
214
nm |
| Flow rate: |
As indicated |
| Gradient |
As indicated |
| Temperature |
48
°C
for digestion. RT for separation. |
| Injection volume |
10
m
l |
| Sample: |
10 mg/ml
Cytochrome c in mobile phase buffer A. |
| 
