Waters Enzymate BEH Pepsin Columns - Manual (Page 2)

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[ CARE AND USE MANUAL ]
Enzymate BEH Pepsin Column
2
Protein in H O, pH 7.0, 20 °C
Protein Labeling
Labeling Quenched
Enzymate BEH Pepsin Column
UPLC Separation
Add excess D
2
O
Reduce pH to 2.5
ESI MS Analysis
Global analysis Local analysis
Maintained at
10–20 ˚C
HDX Manager
II. HOW WAT ERS HDX T ECHNOLOGY WORKS
Waters offers a complete HDX MS system for protein higher-order
structure analysis, such as protein conformational dynamics,
protein-drug binding, protein-protein interactions including
epitope mapping, and protein aggregation. This integrated system
includes automated sample preparation with a LEAP system,
on-line pepsin digestion using the Enzymate BEH Pepsin Column,
UPLC, and QTof MS instrumentations for peptide separation
and characterization with automated data processing and
interpretation by DynamX.
ACQUITY UPLC M-Class with HDX Technology workflow:
1. Deuterium oxide (D
2
O) is added to the protein sample which is
solubilized in a physiological buffer (e.g., 100 mM phosphate
buffer, pH 7.0).
2. The exchange reaction proceeds until a defined end point or
multiple time points for rate determinations.
3. The exchange reaction is stopped by reducing the pH to 2.5
and decreasing the temperature to 0 °C.
4. Sample is injected into the injection port of the HDX Manager
which is maintained at 0 °C.
5. Proteins are digested into peptides on the Enzymate BEH
Pepsin column maintained at 10–20 °C.
Figure 1. How Hydrogen Deuterium Exchange (HDX) works in the ACQUITY UPLC M-Class System with HDX Technology.
6. The peptides are then trapped on a VanGuard™ Pre-Column.
7. The trapped peptides are then eluted from the trap column,
and separated on an ACQUITY UPLC Column at 0 °C.
III. GET TING START ED
a. Column Connection
The Enzymate BEH Pepsin Column uses eXtended Performance
[XP] or ACQUITY UPLC Column-style hardware. Therefore, the
internal geometry of the Enzymate BEH Pepsin Column end nuts
is the same as eXtended Performance [XP] or ACQUITY UPLC
Columns. This geometry is very similar to “Parker-style” depths
which is the LC industry standard. It is important, however, to
reset the fitting every time a new Enzymate BEH Pepsin Column is
installed to ensure no voids or leaks occur.
Use 1/16” PEEK tubing (0.005” internal diameter) for the
Enzymate BEH Pepsin Column inlet and outlet tubing, hand cut
to the appropriate lengths (e.g., part number WAT022995).
Alternatively, 1/16” steel tubing of similar dimensions and
lengths may also be used.