Waters Enzymate BEH Pepsin Columns User Manual

[ CARE AND USE MANUAL ]

Enzymate BEH Pepsin Column

CONTENTS

I. INTRODUCTION

II. HOW WATERS HDX TECHNOLOGY WORKS

III. GETTING STARTED

a. Column Connection

b. Column Installation and Equilibration

c. Digesting/Trapping Flow Rate

d. Quenc hing

e. Protein Injection

f. Trapping Time

g. pH and Solvent Compatibility

h. Temperature

i. Column Washing/Cleaning

j. Column Storage

IV. CHECKING FOR ENZYMATIC ACTIVITY

V. REFERENCES

I. INTRODUCTION

Thank you for choosing the Waters Enzymate™ BEH Pepsin Column.

Pepsin is immobilized onto rugged 5 μm BEH (Ethylene Bridged

Hybrid) particles which are packed into low dispersion 2.1 x 30 mm

stainless steel column hardware containing 0.5 μm porosity inlet

and outlet frits. This is the same column hardware used in our

eX tended P erformance [XP] Column line. This online, flow-through

digestion column reproducibly digests intact proteins into peptides

after incubating with H2O/D2O during HDX experiments. Benefits

of online protein digestion include reduced sample preparation

time, reproducible digests, minimized back-exchange, and limited

enzyme in solution.

The Enzymate BEH Pepsin Column is housed in a temperature-

controlled compartment to ensure optimal performance and

reproducibility. Once the peptic peptides elute from the pepsin

column, they are focused on a trap column. The peptides are then

separated at 0 ˚C on an ACQUITY UPLC® BEH C18 Column and

detected by high resolution mass spectrometry.

The Enzymate BEH Pepsin Column was designed and tested

specifically for use on the ACQUITY UPLC M-Class System with

HDX Technology and the nanoACQUITY UPLC® System with HDX

Technology. Waters cannot support the use of the Enzymate BEH

Pepsin Column on any other LC system.

[ CARE AND USE MANUAL ]

2

Protein in HO, pH 7.0, 20 °C

Protein Labeling

Labeling Quenched

Enzymate BEH Pepsin Column

UPLC Separation

Add excess D2O

Reduce pH to 2.5

ESI MS Analysis

Global analysisLocal analysis

Maintained at

10–20 ˚C

HDX Manager

II. HOW WATERS HDX TECHNOLOGY WORKS

Figure 1. How Hydrogen Deuterium Exchange (HDX) works in the ACQUITY UPLC M-Class System with HDX Technology.

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

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.

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,

III. GETTING STARTED

UPLC, and QTof MS instrumentations for peptide separation

and characterization with automated data processing and

interpretation by DynamX.

a. Column Connection

The Enzymate BEH Pepsin Column uses eXtended Performance

[XP] or ACQUITY UPLC Column-style hardware. Therefore, the

ACQUIT Y UPLC M-Class with HDX Technology workflow:

1. Deuterium oxide (D2O) 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.

internal geometry of the Enzymate BEH Pepsin Column end nuts

is the same as eX tended 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.

5. Proteins are digested into peptides on the Enzymate BEH

Pepsin column maintained at 10–20 °C.

Enzymate BEH Pepsin Column

2